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SHOTGUN LIPIDOMICS: ELECTROSPRAY IONIZATION

MASS SPECTROMETRIC ANALYSIS AND QUANTITATION


OF CELLULAR LIPIDOMES DIRECTLY FROM CRUDE
EXTRACTS OF BIOLOGICAL SAMPLES

Author Proof

A
Xianlin Han*1,2 and Richard W. Gross1,2,3,4
1
Division of Bioorganic Chemistry and Molecular Pharmacology,
Washington University School of Medicine, St. Louis, Missouri 63110
2
Department of Medicine, Washington University School of Medicine,
St. Louis, Missouri 63110
3
Department of Molecular Biology & Pharmacology, Washington University
School of Medicine, St. Louis, Missouri 63110
4
Department of Chemistry, Washington University School of Medicine,
St. Louis, Missouri 63110

Received 25 January 2004; revised 15 March 2004; accepted 15 March 2004

Published online 00 Month 2004 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.00000

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
II. Classification of Cellular Lipids and Organization
A. Non-Polar Lipids . . . . . . . . . . . . . . . . . . .
B. Polar Lipids . . . . . . . . . . . . . . . . . . . . . . .
1. Phospholipids . . . . . . . . . . . . . . . . . . .
2. Sphingolipids . . . . . . . . . . . . . . . . . . .
3. Glycolipids . . . . . . . . . . . . . . . . . . . .
C. Metabolites . . . . . . . . . . . . . . . . . . . . . . .

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III. Principles of ESI/MS of Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00


A. Principles of Electrospray Ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
B. Classification of Cellular Lipids Based on Their Electrical Propensities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
IV. Intrasource Separation for Lipidome Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Intrasource Separation Strategy for Lipidome Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Analyses of Anionic Lipid Molecular Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Analyses of Weak Anionic Lipid Molecular Species . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Analyses of Electrically Neutral but Polar Lipid Molecular Species . . . . . . . . . . . . . . .
4. Analyses of Specialized Lipids and Lipid Metabolites . . . . . . . . . . . . . . . . . . . . . . . . .
B. Technical Aspects Facilitating the Successful Performance of ESI/MS Employing Intrasource
Separation of Lipid Classes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Lipid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Lipid Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Internal Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Instrument Parameters for Shotgun ESI/MS of Lipids . . . . . . . . . . . . . . . . . . . . . . .

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V. Quantitation of Cellular Lipidomes by ESI/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00


VI. Comparisons between Different ESI/MS Approaches to the Global Quantitative Analyses of Cellular Lipidomes . . . . 00
A. Quantitation of Cellular Lipidome by the Intrasource Separation Approach of ESI/MS . . . . . . . . . . . . . . . . . . . 00
B. Quantitation of Cellular Lipidome by Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

*Correspondence to: Xianlin Han, Division of Bioorganic Chemistry


and Molecular Pharmacology, Department of Medicine, Washington
University School of Medicine, Box 8020, 660 South Euclid Avenue,
St. Louis, MO 63110. E-mail: xianlin@wustl.edu
Contract grant sponsor: NIH; Contract grant numbers P01HL57278,
R01HL41250, P41-RR00954, P60-DK20579, P30-DK56341.

Mass Spectrometry Reviews, 2004, 23, 1 53


# 2004 by Wiley Periodicals, Inc.

MAS-490

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HAN AND GROSS

Author Proof

C. Quantitation of Cellular Lipidome by FTMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00


D. Quantitation of Cellular Lipidome by a HPLC-ESI/MS Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
VII. Examples of Lipidome Analysis under Normal and Pathological Conditions . . . . . . . . . . . . . . . . . . . . . . . . .
A. Alterations in Individual Phospholipid Molecular Species of Human Platelets during Thrombin Stimulation
B. Lipid Alterations in Diabetic Myocardium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Specific Lipid Changes in the Very Early Stages of Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . .
D. The Role of Phospholipase Da in Plant Lipid Alterations under Freezing Stresses . . . . . . . . . . . . . . . . . .
E. Alterations in Phospholipids in Mast Cells during Degranulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
F. Analysis of Phospholipid Molecular Species Directly form Cellular Lipid Extracts by HPLC-ESI/MS . . . .

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VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

LipidomicsQ1, after genomics and proteomics, is a newly and


rapidly expanding research field that studies cellular lipidomes
and the organizational hierarchy of lipid and protein constituents
mediating life processes. Lipidomics is greatly facilitated by
recent advances in, and novel applications of, electrospray
ionization mass spectrometry (ESI/MS). In this study, we will
focus on the advances in ESI/MS, which have facilitated the
development of shotgun lipidomics and the utility of intrasource
separation as an enabling strategy for utilization of 2D mass
spectrometry in shotgun lipidomics of biological samples. The
principles and experimental details of the intrasource separation
approach will be extensively discussed. Other ESI/MS approaches towards the quantitative analyses of global cellular
lipidomes directly from crude lipid extracts of biological samples
will also be reviewed and compared. Multiple examples of
lipidomic analyses from crude lipid extracts employing these
approaches will be given to show the power of ESI/MS techniques
in lipidomics. Currently, modern society is plagued by the sequelae of lipid-related diseases. It is our hope that the integration of
these advances in multiple disciplines will catalyze the development of lipidomics and such development will lead to improvements in diagnostics and therapeutics, which will ultimately
result in the extended longevity and an improved quality of life
for humankind. # 2004 Wiley Periodicals, Inc., Mass Spec Rev
9999:153, 2004
Keywords: Alzheimers disease; diabetes; lipidome; lipidomics; electrospray ionization mass spectrometry

I. INTRODUCTION

Lipids are essential cellular constituents that have multiple


distinct yet critical roles in cellular function. First, the
majority (by mass) of cellular lipids form a membrane
bilayer whose integrity and physical characteristics are
vital for life processes. Second, lipids provide an appropriate hydrophobic environment for membrane protein
function and interactions. Third, cellular lipids serve as reservoirs for energy storage, which can be rapidly accessed
2

at times of demand. Finally, of course, membrane lipids are


the source of lipid second messengers generated by the
actions of a variety of intracellular enzymes (Spector &
Yorek, 1985; Ghosh, Strum, & Bell, 1997; Alessenko &
Burlakova, 2002; Roberts, 2002). The diversity of lipids
that participate in signaling functions is underscored by
the multiple integrated roles of eicosanoids, lysolipids,
diacylglycerols (DAG), ceramides, phosphatidic acids, and
others in cellular signaling. Each covalent entity has an
important yet specific role in the cellular signal transduction (Gross, 1992; Di Marzo, 1995; Khan, Blobe, &
Hannun, 1995; Moolenaar, 1995; Bocckino & Exton,
1996; English, 1996; Meade et al., 1996; Ashcroft, 1997;
Athenstaedt & Daum, 1999; Goni & Alonso, 1999;
Lennartz, 1999; Hannun & Luberto, 2000; Kolesnick,
Goni, & Alonso, 2000; Merrill, 2002). For example,
plasmenylethanolamine (PlsEtn) is a subclass of ethanolamine glycerophospholipid (PE), which are highly
enriched in electrically active tissues (including cardiac
subcellular membranes and neuronal cell membranes)
(Gross, 1984, 1985; Han et al., 2001Q2). These phospholipid molecular species play important roles as reservoirs
for arachidonic acid (AA) release, facilitate membrane
fusion, and protect cells from the effects of oxidation
(Zoeller, Morand, & Raetz, 1988; Scherrer & Gross, 1989;
Glaser & Gross, 1994, 1995; Murphy, 2001). Specific
cellular TAG molecular species facilitate energy storage in
living organisms, but in excess are also a factor contributing to atherosclerosis and myocardial dysfunction
(Han et al., 2000; Stanley, 2001; Unger & Orci, 2001;
Unger, 2002).
Thousands of individual lipid molecular species are
present in cells, each interacting in different compartments
and within each bilayer compartment. In addition, many
laterally segregated domains are present, which facilitate
specific interactions and cellular function [e.g., caveolae
(Pike, 2003)]. Moreover, nearest neighbor interactions

SHOTGUN LIPIDOMICS BY ESI/MS

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(annular lipids) dramatically affect transmembrane protein


kinetics and function using membrane dynamics to exploit
basic principles of physical chemistry, which regulate
cellular physiologic function and adaptation. In most
mammalian cells, phospholipids (a phosphodiester linked
to the sn-3 position of glycerides) account for approximately 60 mol% of total lipids; glycolipids/sphingolipids
(carbohydrate(s) linked to the sn-3 position of diglycerides
or the position 1 of the sphingosine) are approximately
10 mol% of total lipids; non-polar lipids (including TAG
and cholesterol) range from 0.1 to 40 mol% depending on
cell type and subcellular compartment. Metabolites (such
as NEFA, lysolipids, DAG, ceramides, acyl carnitines, acyl
CoA) typically represent less than 5 mol% of total cellular
lipids but during pathologic conditions can accumulate and
contribute to deleterious pathophysiologic sequelae.
The precise complement of chemically distinct
covalent entities in cellular lipids has been referred to as
the cellular lipidome. Research in lipidomics examines
alterations in the interactions of constituents in the cellular
lipidome using multiple different techniques. Thus, investigators in lipidomics must first quantify the thousands of
chemically distinct constituents present in the cellular
lipidome, determine their subcellular organization (subcellular membrane compartments and microdomains),
identify kinetic fluxes into and out of each cellular compartment, and delineate lipidlipid and lipidprotein
interactions, which collectively determine membrane conformational space and dynamics. The first step in global
lipidomics is to measure the amount of each distinct
chemical entity present in a cells lipidome and identify
those alterations in chemical constituents that precipitate,
or are associated with, phenotypic alterations. Although
lipidomics is a newly expanding field, its foundations and
importance in disease states have been rooted in the
literature for over 20 years (Gross, 1984, 1985). The study
of cellular lipidomes has already provided many new
insights into disease states through the detailed quantification of alterations in a cells lipidome (e.g., lipid classes,
subclasses, and individual molecular species) with disease
and examination of the kinetics of lipid metabolism and
the interactions of lipids with cellular proteomes (e.g.,
Hazen et al., 1993; Han et al., 1996, 2000, 2001, 2002).
As a field, lipidomics has lagged in comparison to
the development of genomics and proteomics. One of
the reasons underlying the delay of our understanding
of lipidomics has been the lack of analytical techniques
sensitive enough for lipidome analyses. However, lipidomics is greatly facilitated by recent advances in, and novel
applications of, electrospray ionization mass spectrometry
(ESI/MS) and tandem mass spectrometry (MS/MS) that
have very recently been reviewed by Griffiths (2003), Han
& Gross (2003), Pulfer & Murphy, (2003), and Welti &
Wang (2004). Griffiths (2003) has extensively covered the

tandem mass spectrometric characterization of fatty acids,


bile acids, and steroids as well as the studies on complex
lipids such as phospholipids and triacylglycerols (TAGs) to
some degree. Pulfer & Murphy (2003) have focused on
the detailed characterization of each of the phospholipid
classes. Welti & Wang (2004) have focused on the direct
profiling of lipids from plants. We have briefly discussed
the utility of intrasource separation for the application of
global analyses of cellular lipidomes directly from crude
extracts of biological samples (Han & Gross, 2003). This
study will focus on the detailed developments necessary
for shotgun lipidomics by ESI mass spectrometry and the
utility of intrasource separation 2D mass spectrometric
approaches in shotgun lipidomics. The principles and
experimental details of intrasource separation approach
will be extensively discussed. Other ESI/MS approaches for the quantitative analyses of global cellular
lipidomes directly from crude lipid extracts of biological
samples will also be discussed and mutually compared.
Multiple examples of global lipid analyses from crude lipid
extracts employing these approaches will be given to
show the power of ESI mass spectrometric techniques in
lipidomics.
We would like to stress that the shotgun lipidomics
based on intrasource separation reviewed herein only
represents one approach for global lipidome analysis.
Although over 90% of lipid mass has been accounted for
and quantitated by this approach directly from the lipid
extracts of biological samples, thousands of very low
abundance lipid molecular species are not approachable
by this technique at its current stage of development. For
those molecules, many analytical methods already exist to
enrich low abundance lipid molecular species, which can
be integrated into the current platform. Of course, many
new methods still need to be developed and tailored for
thorough analysis of the cellular lipidome. Collectively,
shotgun lipidomics is still in its early stage but even today it
provides a strong foundation for the analysis of thousands
of molecular species after data are judiciously analyzed by
bioinformatic processes. It is our sincere hope that the
integration of the rapid advances in all developed and/
or developing approaches will catalyze the development
of lipidomics, which will lead to the improvement of
diagnostics and therapeutics and the extended longevity in
humankind.

A
II. CLASSIFICATION OF CELLULAR LIPIDS
AND ORGANIZATION

As the smallest self-renewing unit of human life, mammalian cells contain multiple subcellular organelles (e.g.,
plasma membranes, mitochondria, Golgi, endoplasmic
reticulum, and peroxisomes), which each performs specific
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functions. Microdomains are present in each subcellular


membrane (e.g., caveolae and lipid rafts present in plasma
membranes), which further underscore the highly defined and regulated structures of mammalian membranes.
Mammalian membranes are comprised of proteins and
lipid constituents. Membrane proteins account for approximately 70% of membrane mass, whereas lipids represent
the remainder. However, the typical weight of the membrane protein is approximately 80 kD. Accordingly, there
are typically 10100 lipid molecules (or more) for every
protein depending on the subcellular organelle studied.
Obviously, the ratio and organization is critical for appropriate biological function. Biological membranes are
organized into bilayers by hydrophobic forces and contain
an astonishing diversity of lipids (in the thousands to tens
of thousands). These lipids posses a unifying characteristic
(i.e., their amphipathic character), which results from the
presence of a polar or hydrophilic head group and a nonpolar or hydrophobic region which serves to distinguish
lipid physical characteristics, thereby modulating biological functions. It is this critical integration of form and
function that has fascinated membrane chemists for nearly
a century.
As discussed, cellular lipids are not homogeneously
distributed in each cellular membranes, but rather each
membrane is specifically tailored in microdomains in each
subcellular membrane compartment. The lipid composition of two leaflets of membrane bilayers are also very
different. Choline glycerophospholipid (PC), sphingomyelin (SM), and cholesterol are mainly located in laminale
leaflet of the membrane bilayer, whereas PE, phosphatidylinositol (PtdIns), and phosphatidylserine (PtdSer) are
mainly resided in the cytosolic leaflet of the membrane.
Collectively, the presence of multiple subcellular organelles and membrane microdomains, the heterogeneous
distribution of cellular lipids, as well as the inherent
dynamic nature of lipid interactions makes the research in
lipidomics both fascinating and difficult. Accordingly,
lipidomics has become a new rising and spreading research
field, which integrates genomics and proteomics with
human disease processes. Cellular lipids can be roughly
classified into three large groups including non-polar
lipids, polar lipids, and metabolites based on their relative
polarities of the head group regions and/or their relative
mass content.

A. Non-Polar Lipids

Non-polar lipids include predominantly (by mass) cholesterol and cholesterol esters (Fig. 1A,B) as well as TAGs
(Fig. 1C) which vary greatly accordingly to the cell type
and subcellular fraction examined. All these classes of
lipids consist of a small or weakly polar part and a dominant
hydrophobic region. Cholesterol mass levels in different
4

FIGURE 1. The general structures of cholesterol (A), cholesterol ester

(B), and triacylglycerol (C, TAG). R, R1, R2, and R3 represent aliphatic
chains usually containing 1323 of carbons and 06 of double bonds for
naturally occurring compounds.

membrane compartments and/or cell type can vary from


near 0 to as much as 40 mol% of mammalian plasma
membrane lipids (Pak, Han, & Gross, 1992; Cullis, Fenske,
& Hope, 1996), which have dramatic effects on membrane
physical properties (Kimelberg, 1985; Presti, 1985).
Cholesterol esters and TAGs are mainly present in the
cellular oil droplets and lipoproteins, whereas nonesterified
cholesterol is mainly present in plasma membranes.

B. Polar Lipids

1. Phospholipids

Polar lipids are predominately comprised of phospholipids, sphingolipids, and glycolipids. The glycerol-based
phospholipids are predominant in eukaryotic cells, accounting for approximately 60 mol% of lipid mass. The
distinguishing characteristic of phospholipids is the presence of at least one phosphate group at the sn-3 position of
glycerol backbone (Fig. 2A). Based on the differences in
the chemical structures (X) that are linked to the phosphate,
phospholipids can be classified into multiple different
classes (Fig. 2B). For example, if X is choline or ethanolamine (Fig. 2B), the corresponding class of the phospholipids is PC or PE, respectively. Moreover, there are
multiple subclasses present in these two classes of phospholipids (see below), which have important effects on

SHOTGUN LIPIDOMICS BY ESI/MS

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A
FIGURE 2. The general structure and classes of phospholipids. The classification of phospholipid classes is

based on the moieties of X that is a part of a small molecule less than a hydroxyl group.

cellular function and lipid second messenger generation.


Other classes of phospholipids include phosphatidylglycerol (PtdGro), PtdIns, PtdSer, diphosphatidylglycerol
(or cardiolipin), etc. (Fig. 2B). Subclass counterparts of
diacyl phospholipids in these latter classes are modest
in mammals. Therefore, subclasses of these particular
phospholipid classes are not further discussed in this study
and attention will focus on the subclass diversity in choline
and PEs. In most eukaryotic membranes, PC and PE are
dominant, present in an approximately 3:2 molar ratio and
account collectively for approximately 75 mol% of total
phospholipid mass. The other classes of phospholipids are
present in approximately 20 mol% of phospholipid mass
and vary from a few percent to over 10 mol% depending on
the cell type and subcellular membrane compartment.
However, in most prokaryotic cells, PC is not usually
present, whereas PE, PtdGro, and cardiolipin are the major
classes of phospholipids (Rock, Jackowski, & Cronan,
1996). Phospholipids usually distribute asymmetrically in
the membranes. For example, PC is dominant in the outer
monolayer and PE and PtdSer are mainly present in the
inner monolayer of plasma membranes (Cullis, Fenske, &
Hope, 1996).
Based on the covalent nature of the linkage of the
aliphatic chain at sn-1 position of glycerol backbone, each
phospholipid class is further subdivided into three subclasses, i.e., phosphatidyl, plasmenyl, and plasmanyl,
corresponding to ester, vinyl ether, and alkyl ether linkages,
respectively (Fig. 3). However, these alternative sub-

classes are found mainly in PE and PC classes (Horrocks &


Sharma, 1982). In most cellular membrane lipids, the
phosphatidyl subclass of phospholipids is predominant.
However, in most electroactive cellular membranes, such
as sarcolemma and neuronal cells, plasmenyl subclass
molecular species are the major components of cellular
lipids (Gross, 1984, 1985; Han & Gross, 2001). The plasmenyl subclasses of other phospholipid classes typically
only constitutes a very minor component in other cells with
the exception of plasmenylserine that is abundant in some
types of fishes (Horrocks & Sharma, 1982) and is also
present in nontrace quantities in mammalian cells.
The complexity of phospholipid molecular species is
not only because of the variation of polar head groups
(classes) and the different linkages of sn-1 aliphatic chains
(subclasses), but also because of the chain length as well as
the number and location of double bonds of two aliphatic
chains in phospholipid molecule. Thousands of molecular
species of phospholipids can readily be constructed in
theory, and over five hundreds of phospholipid molecular
species have been demonstrated from mouse B cell
myeloma NS-1 cells in practice (Taguchi et al., 2000).
2. Sphingolipids

The second major category of polar lipids are sphingolipids that contain a sphingosine (i.e., trans-4-sphingenine)
backbone or its analogs and represent approximately 5
10 mol% of total lipid mass in most brain cells. Remark5

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A
FIGURE 3. The subclasses of ethanolamine glycerophospholipids that are classified based on the linkage of

aliphatic chain at the sn-1 position of glycerol backbone. The subclasses of other phospholipid classes are
identically classified and abbreviated in consequence.

ably, sphingolipids account for over 30 mol% of total lipids


present in human white matter (Han et al., 2002). Based on
the nature of the covalent linkage of the polar moieties to
ceramide (i.e., N-acylsphingosine), sphingolipids can be
classified into SM, cerebroside, glucosylceramide, lactosylceramide, sulfatide, and other glycosphingolipids that
contain multiple sugar rings (Fig. 4). More detailed
structures and their nomenclatures of these glycosphingolipids that contain multiple sugar rings can be found in
prior reviews (e.g., Merrill & Sweeley, 1996). Although the
sphingosine (18-carbon homolog) backbone is the most
prevalent in most mammalian sphingolipids, sphingolipids
with other sphingosine analogs that contain alkyl chain
lengths from 14 to 22 carbon atoms are also present in some
cases and in other species. For example, tetradeca-4- and/
or hexadeca-4-sphingenine backbones are dominant in
sphingolipids in Drosophila (Sugita et al., 1989; Rietveld
et al., 1999; Acharya et al., 2003). In addition to trans-4sphingenine, saturated sphinganine, 4-D-hydroxysphinganine, and branching sphingosine are also common (Merrill
& Sweeley, 1996).
3. Glycolipids

The third major category of polar lipids is the glycolipids,


which include glycosphingolipids and glycoglycerolipids.
The main difference between these two classes of glycolipids is the hydrophobic core. The former contains a
ceramide backbone (see last paragraph) and the later
6

FIGURE 4. The general structure and classes of sphingolipids. The

classification of sphingolipid classes is based on the moieties of X. Glc,


Gal, and Neu5Ac represent glucose, galactose, and N-acetylneuraminic
acid (i.e., sialic acid), respectively.

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contains a DAG backbone. More detailed structures and


their nomenclatures of glycoglycerolipids can be found in
the chapter written by Ishizuka & Yamakawa (1985).
In some cells, glycolipids account for up to approximately
10 mol% of total lipid mass.

broad range of compounds. Many theories of the physical


processes involved in ion generation during ESI have been
proposed and validated in some detail (Fenn et al., 1990;
Smith et al., 1990; Cole, 1997; de la Mora et al., 2000).
The principles of the ESI process can be schematically
diagrammed as shown in Figure 5. Initially, a solution
containing the analytes of interest is introduced into the
ESI ion source through capillary tubing. The narrowed
orifice at the end of the capillary tubing and the mechanical
forces imparted as the solution passes through the narrow
orifice facilitates the formation of sprayed small droplets in
the ionization chamber. If an electric potential (approximately 4 kV) is applied between the end of the capillary
tube and the entry into the mass analyzer, because of
oxidation/reduction processes, the droplets can carry net
charges and are directed into the mass analyzer by the
applied electric field. As shown in Figure 5, if a positive
electric potential is applied to the end of capillary tube and
a negative electric potential is present at the entrance of the
mass analyzer in the positive-ion mode, droplets carry net
positive charges. During flight, the droplets are desolvated,
either by passage through a curtain of heated inert gases
or alternatively through a heated capillary, or both. Thus,
during desolvation, the coulombic force between ions is
dramatically increased. Once this force exceeds the surface
tension of the solvent, the droplets explode to form smaller
droplets. This cycle is iteratively repeated until molecular
ions are generated prior to their entrance into the mass
analyzer. Although many physicochemical features of the
ionization and fragmentation process are still unclear,
droplet surface tension and the spatial proximity of surface

C. Metabolites

The last group of lipids are lipid metabolites, which are


derived by enzymatic action of parent lipids on their
precursors. Metabolites can either be anabolic or catabolic
in nature. Although this group of lipids are only a few
percent of total lipid mass under normal physiological
conditions, the numbers of molecular species are substantial. Common metabolites include long chain acylCoAs, long chain acylcarnitines, nonesterified fatty acids
(NEFA), fatty acid esters, acyl anamide, ceramides,
lysolipids, eicosanoids, DAGs, sphingosine-3-phosphate,
etc. Many of the metabolites are biologically active second messengers. Moreover, in some cases (e.g., cardiac
ischemia), lipid metabolites constitute substantial fractional percentages of lipids in some compartments and
have been implicated in many disease states (Han & Gross,
1991).
III. PRINCIPLES OF ESI/MS OF LIPIDS
A. Principles of Electrospray Ionization

ESI/MS, initially developed by Fenn et al. (1989), has been


extensively used in many applications for the analysis of a

FIGURE 5. Schematic diagram of the principle of electrospray ionization in positive-ion mode.

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charges on sprayed droplets are critical determinants of the


ionization process.
The ionization efficiency of an analyte greatly depends
on the electrical propensity of an individual analyte in
its own microenvironment to lose or gain a charge (i.e.,
oxidize or reduce) in an electric field. Figure 6 illustrates
the principles of the relationship between ion formation,
ionization efficiency, and the electrical properties of
specific analytes. Either the cationic or the anionic region
in compounds that possess bonds with easily separable
charges can be easily ionized in either positive- or negativeion modes of ESI, respectively. Because these cationic and
anionic moieties carry inherent charges that can be readily
separated in the ESI ion source, the ionization efficiency
for these ions is much higher relative to that for compounds
that do not carry an inherent charge. Although an intrinsic
ionizable site may be absent in a given compound,
ionization can also be achieved in the ESI ion source
through formation of adduct ions. Provided a sufficient
dipole potential is present, an analyte can be induced to
interact with either a small anion or cation in the media
facilitating ionization in the ESI ion source and thus is
potentially suitable for analysis by ESI/MS and ESI/
MS/MS. The ionization efficiency of different covalently
linked compounds depends on the dipole potentials present
in each analyte.
For example, PC molecular species do not carry a net
charge but contain a large dipole in the zwitterionic polar
head group. Thus, PC molecular species can form adducts
either with proton ([M H]), lithium ([M Li]),
sodium ([M Na]) in the positive-ion mode (Duffin,
Henion, & Shieh, 1991; Weintraub, Pinckard, & Hail,
1991; Han & Gross, 1994; Kerwin, Tuininga, & Ericsson,

1994; Kim, Wang, & Ma, 1994; Hsu, Bohrer, & Turk,
1998b), or with chloride ([M Cl]), acetate ([M
OAc]) in the negative-ion mode (Kerwin, Tuininga, &
Ericsson, 1994; Han & Gross, 1995). The abundance of
the cation or anion adducts is dependent on the concentration of the cations or anions in solution and the affinity
of these cations or anions for the zwitterionic compounds
of interest. It is well known that the head group component of PC molecular species contributes the dominant
dipole of the molecules and other dipole components are
small by comparison. Therefore, the ionization efficiency
of different naturally occurring PC molecular species is
essentially identical within experimental error after
correction for 13C isotope effects under conditions utilizing
very dilute solution of lipids (Han & Gross, 1994, 2003)
(vide infra).
Although TAGs are normally termed non-polar lipids,
adduct ions of TAG can be formed with ammonium,
lithium, or sodium ions with remarkable sensitivity in the
femtomole range (Duffin, Henion, & Shieh, 1991; Hsu &
Turk, 1999; Han et al., 2000; Han & Gross, 2001). In
contrast to PC molecular species, a dominant dipole component in TAG molecules is absent and thus alterations in
the acyl chain length and the numbers of double bonds
affect the overall dipole of the TAG molecule. Accordingly,
the ionization efficiency of different TAG molecular
species is substantially different, as demonstrated by the
correlation of the ionization efficiency to the total carbon
number and the number of double bonds (Han & Gross,
2001).
REVIEW THESE CITED PAPERS

FIGURE 6. The relationship of ion formation and ionization efficiency

with the electrical propensity of an analyte. The adduct ion X or Y for a


covalently-linked polar compound depends on the availability of small
cation or anion in the solution and the affinity of the adduct ion with the
polar compound.

B. Classification of Cellular Lipids Based on


Their Electrical Propensities

Cellular lipids are typically grouped into many different


classes based on the covalent nature of their polar head
groups and/or the backbones linked to the aliphatic chains
(see section II). These groupings lead to the consideration
of four different categories of lipids based on their electrical
propensities (Table 1), which have been exploited for ESI/
MS analyses of lipids (Fig. 6). The first category of cellular
lipids are anionic lipids that contain one or more net
negative charge(s) at physiological (neutral) pH. The major
classes of anionic lipids include cardiolipin, PtdGro, PtdIns
(PtdInsP, PtdInsP2), PtdSer, PtdH, acylCoA, sulfatide,
cholesterol sulfate, etc.
The second category of cellular lipids we consider are
weak anionic lipids that carry a net negative charge only
after they are induced to form their conjugate acid at
alkaline pH. Although they tend to yield negative ions in
negative-ion mode under physiological pH, their ionization efficiency is substantially lower compared to lipids in
the first category. Lipids in this category commonly include
moieties containing an ethanolamine head group (e.g., PE

SHOTGUN LIPIDOMICS BY ESI/MS

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TABLE 1. Classification of lipid classes based on their electrical propensities

and lysoPE), NEFA and some of their derivatives such as


eicosanoids, bile acids, etc. The third category of cellular
lipids are polar but electrically neutral lipids at both physiological and alkaline pH conditions. This group of lipids
include choline-containing lipids (such as PC, lysoPC, and
SM), glycolipids containing non-zwitterionic polar head
group (e.g., cerebrosides), TAG, DAGs, etc. The fourth
category of lipids are some specialized lipids such as
long-chain acylcarnitines, cholesterol and its derivatives
including cholesterol esters and sterols, etc.

analysis of cellular lipidome directly from crude extracts of


biological samples. In addition, through judicious additions of appropriate ions, analysis of some specialized lipid
classes (e.g., acylcarnitines) can be performed directly
from a lipid extract (see below).
1. Analyses of Anionic Lipid Molecular Species

As previously discussed, anionic lipids that possess an


inherent negative charge or charges in the chloroform
extract of a biological sample can be analyzed by ESI/MS

IV. INTRASOURCE SEPARATION


FOR LIPIDOME ANALYSES
A. Intrasource Separation Strategy
for Lipidome Analyses

Given the principles of the ESI process and the relationship


between ionization efficiency and the electrical propensity
of cellular lipids in the context of their distinct covalent
structures, the stage was set to perform total cellular
lipidome analyses by ESI mass spectrometric techniques
directly from crude extracts of biological samples without
prior chromatographic separation. Founded on these basic
chemical principles, a strategy for lipidome analyses of
biological samples in combination with the appropriate
front-end sample preparation (i.e., pH adjustment) without
chromatographic separation (Fig. 7) has been developed
and hereafter referred to as intrasource separation (Han &
Gross, 1994, 2003). Using this strategy, direct fingerprinting and quantitation of hundreds of individual lipid
molecular species including most of the major lipid classes
and many minor metabolite groups (Han & Gross, 2003)
has been possible. This strategy provides a platform for

FIGURE 7. Schematic diagram of the intrasource separation strategy

used for global lipidome analyses directly from a crude extract of a


biological sample.

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at neutral pH. For example, the ESI mass spectrum of a


crude lipid extract from mouse myocardium displays
abundant deprotonated anionic lipid molecular ions such as
PtdIns, PtdGro, PtdSer, cardiolipin, and PtdH (Fig. 8A),
whereas it only shows low abundant pseudomolecular
ions from other classes of lipids such as PE and PC. For
example, the amount of internal standard for some of the
anionic lipids (i.e., 15:0-15:0 PtdGro, m/z 693.6) added to
the lipid extract is only one-eighth of the amounts of the
internal standards for PE (i.e., 15:0-15:0 PtdEtn, m/z 662.6)
and PC (i.e., 14:1-14:1 PtdCho, m/z 708.6 as a chloride
adduct), but the peak intensity of 15:0-15:0 PtdGro molecular ion is 5- and 20-folds more intense than those of these
PE and PC pseudomolecular ions, respectively (Fig. 8A).
These results demonstrated the selective ionization of
anionic lipids under the conditions.
Ion peaks corresponding to PtdIns molecular species
can be identified by product-ion analysis (Han & Gross,
1995; Hsu & Turk, 2000) or precursor-ion (PI) scanning of
m/z 241.1 corresponding to phosphoinositol (Brugger
et al., 1997; Hsu & Turk, 2000). Ion peaks corresponding
to PtdSer molecular species can be identified by neutral
loss (NL) scanning of 87.0 amu (corresponding to the loss
of serine) or individual product-ion analyses (Han & Gross,
1995; Brugger et al., 1997). Similarly, all of deprotonated
anionic phospholipid ions including PtdH, PtdGro, and
cardiolipin can be identified by PI scanning of m/z 153.0
corresponding to glycerophophate (Table 2; Fig. 8B) or
individual product-ion tandem mass spectrometric analyses (Han & Gross, 1995). The acyl moieties, the regiospecificity and the isobaric peak composition can also
be identified and analyzed by PI scanning of all naturallyoccurring fatty acids. Long-chain fatty acyl CoA molecular
species can be analyzed directly from crude lipid extracts
by PI scanning of m/z 339 corresponding to doublycharged CoA group (Kalderon et al., 2002). The classes of
PtdInsP and PtdInsP2 molecular species can be profiled by
PI scanning of m/z 321.1 and 401.1, respectively, directly
from the crude lipid extracts of biological samples as
described previously (Hsu & Turk, 2000; Wenk et al.,
2003). This approach provides a foundation for the quantitative analyses of these classes of molecular species
directly from the crude lipid extracts of biological samples
without prior chromatographic separation. Lyso anionic
phospholipids may be quantitated as described by many
investigators (Baker et al., 2000, 2001; Xiao et al., 2000,
2001; Yoon, Kim, & Cho, 2003). Most of the commonly
used ESI tandem mass spectrometric methods are summarized in Table 2.
Because the electrical propensities of anionic phospholipid classes are predominantly attributable to the
negative charge of the phosphate whereas the effects of
other parts of the molecules are minimal, the ionization
efficiency of anionic phospholipids is nearly identical

within experimental error in dilute lipid solutions (Han &


Gross, 1994). By comparing the intensity of individual
ion peak with an anionic phospholipid internal standard
(or standards) and appropriate correction for 13C isotope
effects (see next section), the mass content of individual
anionic phospholipid molecular species of deprotonated
ions can be obtained from the ESI/MS spectrum under the
conditions. The mass content of low abundant anionic
molecular species can be refined from the analyses of the PI
or NL tandem mass spectra of their head groups.
However, small changes in the pH value of the media
may affect the ionization state of PtdSer and PtdH, which
tend to yield doubly charged ions under physiological
conditions. Thus, internal standards analogous to the
molecular species of these two classes are required for
accurate quantitation of PtdSer and PtdH molecular
species. Cardiolipins are present as both a doubly charged
ion and a minor singly charged ion under the experimental
conditions. In quantitative analysis of cardiolipin, both
ions have to be included and a large 13C correction factor
for both ions need to be considered. Again, small changes
in the pH value of the media may affect the peak intensities
of these ion states, thus, it is better to include a cardiolipin
internal standard for accurate quantitation of cardiolipin.
Cardiolipin molecular species may also be analyzed in
either positive- or negative-ion ESI/MS/MS as recently
described by Beckedorf et al. (2002). It should be noted,
however, that weak anionic lipid molecular species may
also be ionized with less efficiency and polar but electrically neutral lipids may also form small anion adducts
in low abundance under typical experimental conditions
(Fig. 8A). These ions can be easily distinguished from
anionic lipid ion peaks by comparison of the mass spectrum of the identical lipid extract acquired in the presence
of LiOH (Fig. 9, see below).
Sulfatide molecular species are predominantly present
in neuronal and renal tissues and can be profiled by tandem
mass spectrometry by PI scanning of m/z 97.0 corresponding to a sulfate anion (Hsu, Bohrer, & Turk, 1998a;
Table 2). With addition of a synthetic internal standard
(e.g., N16:0 sulfatide) or an anionic internal standard
with a correction factor for ionization efficiency, sulfatide
molecular species can be directly quantitated from crude
lipid extracts as previously described (Han et al., 2002,
2003a; Cheng et al., 2003). Some other sulfated- and
sulfonated lipids can also be analyzed by ESI tandem mass
spectrometry as previously described (e.g., Griffiths et al.,
1996).

10

2. Analyses of Weak Anionic Lipid Molecular Species

By addition of a small amount of weak base (e.g., LiOH


in methanol), weakly anionic lipid molecular species are
rendered anionic in solution by deprotonation of the

SHOTGUN LIPIDOMICS BY ESI/MS

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HOW IS THE SPRAY STABILITY?!
what is IS for PI?

A
FIGURE 8. ESI mass spectrometric analysis of anionic lipids of a crude lipid extract from a mouse

myocardium in negative-ion mode. Panel A: A representative ESI mass spectrum of anionic phospholipid
analyzed directly from a mouse myocardium lipid extract in negative-ion mode. Panel B: A representative
ESI tandem mass spectrometric analysis of anionic phospholipids of a mouse myocardial lipid extract
by precursor-ion (PI) scanning of m/z 153.0 corresponding to a glycerol phosphate derivative. Mouse
myocardial lipids were extracted by a modified Bligh & Dyer (1959) method. The experimental conditions
for PI scanning were listed in Table 2 with a collision gas pressure of 1 mTorr. The identities of all
indicated molecular species have been determined by tandem mass spectrometry. IS represents internal
standard.

11

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TABLE 2. Summary of ESI tandem mass spectrometric methods for lipid analysesa

A
a

The original references from which these methods were employed are cited in the text. See text for the
abbreviations. The method labeled with asterisks (*) may be used for quantitative analysis. The collision energies can
only be used as a reference.

quaternary amine in PE and lysoPE, of the carboxylic


acid in NEFA and eicosanoids, or even of the amide in
ceramides by addition of appropriate amounts of base.
Thus, negative-ion ESI mass spectrometric analysis of
alkaline lipid extracts shows abundant deprotonated ion
peaks corresponding to individual ethanolamine-containing molecular species in the mass range between m/z 600
and 900 for PE (Fig. 9), 400 and 600 for lysoPE, and 200
12

and 400 for NEFA. Note that LiOH, rather than other weak
bases such as NH4OH, is preferable because of the utility
of Li for the ESI/MS analyses of PC, TAG, and other
molecular species as characterized previously (Hsu &
Turk, 1999, 2001, 2002; Han et al., 2000; Han & Gross,
2001).
The acyl chains of ethanolamine-containing lipids
can be identified by PI scanning of all potential naturally

SHOTGUN LIPIDOMICS BY ESI/MS

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A
FIGURE 9. A representative ESI mass spectrum of ethanolamine glycerophospholipid analyzed directly

from a mouse myocardium lipid extract in negative-ion mode in the presence of LiOH. The lipid extract is
identical to the one used for the analysis of anionic phospholipids shown in Figure 8. The identities of all
indicated molecular species have been determined by tandem mass spectrometry. IS represents internal
standard.

occurring acyl carboxylate ions (e.g., m/z 255.2 for 16:0,


279.2 for 18:2, 281.2 for 18:1, 303.3 for 20:4, and 327.3 for
22:6, etc) in a 2D mass spectrometric manner (Fig. 10;
Missing reference!
Han & Gross, 2003). The regiospecificity of the acyl chains
and the composition of molecular species of each isobaric
ion peak can also be substantiated by analyzing the peak
intensity of the acyl carboxylate ions from either their
INDICATE THE
PREDOMINANTproduct-ion or PI spectra (Han & Gross, 1995, 2003).
SPECIES!
LysoPE molecular species can be characterized by production analysis (Han & Gross, 1996). Individual molecular
species of PE and lysoPE in a lipid extract can be quantified
by comparison of their ion peak intensities with that of a PE
or lysoPE internal standard, respectively, after correction
for 13C isotope effects.
There are three subclasses of lipids in PE, which are
distinguished by the nature of the covalent linkage of the
aliphatic chain to the sn-1 position of the glycerol backbone (Fig. 3). PtdEtn can be distinguished from PlsEtn and
the alkyl subclass by ESI tandem mass spectrometry as
discussed above. However, PlsEtn and alkyl subclass can
not be directly discriminated by ESI/MS techniques alone.
Accordingly, to identify ion peaks corresponding to PlsEtn
molecular species, a part of each lipid extract is treated with

acidic vapor prior to mass spectrometric analyses as


described previously (Ford, Rosenbloom, & Gross, 1992;
Kayganich & Murphy, 1992). Through examination of PE
molecular species profiles of both acid-treated and nontreated lipid extracts, PlsEtn molecular species are readily
distinguished from alkyl subclass molecular species. It
should be emphasized that PlsEtn molecular species are
very acid-labile. Thus, acidic conditions have to be avoided
when biological samples containing significant amounts of
molecular species of this subclass are analyzed.
It is also reasonable to consider if anionic lipids in the
extract could overlap with PE molecular species and thus
effect quantitative analyses of PE molecular species. First,
because the mass abundance of PE molecular species is
much higher than that of anionic lipid molecular species
in most of biological samples (see section II and Cullis,
Fenske, & Hope, 1996), the effects of these minor ions on
the quantitation of PE molecular species is within experimental error in most cases (compare Figs. 9 and 8A).
The ion peaks corresponding to anionic lipids including
their internal standard (which are dominantly displayed in
Fig. 8A) are in such low abundance that in most cases these
ions can be neglected in the quantitation of PE molecular
13

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A
FIGURE 10. A two-dimensional ESI mass spectrometric analysis of ethanolamine glycerophospholipid

molecular species in a mouse myocardial lipid extract by PI scanning in negative-ion mode in the presence of
LiOH. All PI scans displayed are normalized to the base peak in the individual scan. From the 2D analyses,
ion peaks can be assigned, isobaric peaks can be identified, and the regiospecificity of each individual
molecular species can be determined.

CHECK THIS!!!

species in Figure 9. Second, PtdSer molecular species


largely form doubly charged molecular ions under the
stated conditions. Therefore, overlap between PE molecular species and PtdSer molecular species is minimal.
Third, because of the naturally occurring differences in
individual PE molecular species and anionic lipid species,
only a few of anionic lipid molecular species in theory
actually overlap with PE species further minimizing errors
from this approach (Figs. 8 and 9). Finally, because of the
presence of a different number of nitrogen atoms in PE
and most of the classes of anionic lipids (one nitrogen
atom is present in PE, whereas no nitrogen atom is present
in PtdGro, PtdIns, PtdH, and cardiolipin), the nitrogen
rule (McLafferty & Turecek, 1993) can be utilized to
deconvolute the partially-overlapped anionic lipid species
14

from PE molecular species by applying correction factors


for 13C isotope effects.
It also should be noted that the abundance of hydroxy(or other small anions such as chloride) associated adducts
of other polar lipids (e.g., PC) is very low (Fig. 9). For
example, ion peaks at m/z 690.6 and 708.6, corresponding
to the hydroxy and chloride adducts of 15:0-15:0 PtdCho
(an internal standard for the quantitation of PC molecular
species, which is very abundant in the lipid extract as
demonstrated in Figure 11), respectively, are absent or
minimal. Moreover, because of the difference of mass
distribution between PE molecular species and PC molecular species adducts, only a few of the PC molecular
species in practice actually overlap with PE species.
Therefore, the effects of these adduct ions on PE analyses

SHOTGUN LIPIDOMICS BY ESI/MS

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A
FIGURE 11. A representative ESI mass spectrum of choline glycerophospholipid analyzed directly from a

mouse myocardium lipid extract in positive-ion mode in the presence of LiOH. The lipid extract is identical
to the one used for the analysis of anionic phospholipids shown in Figure 8. The identities of all indicated
molecular species have been determined by tandem mass spectrometry.

??

can be neglected with errors of 23% in PE quantitation in


extreme cases. Collectively, PE molecular species can be
readily quantitated in negative-ion ESI/MS from a lipid
extract of a biological sample after addition of a small
amount of LiOH with an experimental error of approximately 5%.
Similarly, NEFAs and eicosanoids exist as lithium
coordinated carboxylate anions in solution in the presence
of LiOH. Thus, abundant deprotonated ion peaks corresponding to NEFA molecular species are apparent in the
mass range of m/z 200400 (Han & Gross, 2003). The
NEFA molecular species can be quantitated by comparisons of their ion peak intensities with that of a NEFA
internal standard (e.g., 19:0 FA, 2H31-16:0 FA) after correction for 13C isotope effects. AA can be converted into a
diverse family of biologically active eicosanoid metabolites including prostaglandins, leukotrienes, lipoxins,
hydroxyl fatty acids (HETEs), epoxyeicosatrienoic acids
(EETs), etc. through enzymatic and nonenzymatic reactions. Mass spectrometry has been extensively employed to
characterize and quantitate these large numbered and
complicated compounds (see Murphy, Fiedler, & Hevko,
2001 for recent review). However, majority of the quantitative studies reported are based on strategies using HPLC
techniques. If necessary, ESI mass spectrometric analysis
of these compounds directly from lipid extracts without the

need of chromatographic separation can be further developed since these compounds are susceptible to acidcatalyzed processes, (e.g., oxidation and isomerization)
during sample chromatography. Alternatively, a targeted
lipidomic approach using electron capture atmospheric
chemical ionization mass spectrometry (Lee et al., 2003)
can be employed.
The amide proton [or the proton on other sites (Hsu &
Turk, 2002)] in ceramide molecular species is partially
removed in the presence of LiOH, thus allowing ceramides
to be directly profiled and quantitated by comparisons
with a ceramide internal standard by tandem mass spectrometry as described (Han, 2002). Specifically, three NL
ESI tandem mass spectra under the stated conditions are
acquired for ceramides or its analogs with each type of
sphingoid. For example, for the analysis of ceramides
containing sphingosine (18-carbon homolog) backbone
that is predominant in most mammalian sphingolipids, NL
scanning of 240.2, 256.2, or 327.3 amu can be performed
(Table 2). NL scanning of 256.2 amu (corresponding to
the loss of a water molecule and a 2-trans-palmitoleyl
aldehyde) is selective for nonhydroxy ceramides. NL
scanning of 327.3 amu (corresponding to an acyl carbonyl
anion) is specific for 2-hydroxy ceramides. Thus, all of
ceramide molecular species containing a sphingosine
backbone can be identified from these NL ESI mass
15

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spectra. NL scanning of 240.2 amu (corresponding to the


loss of a 2-trans-palmitoleyl alcohol) results in ion peaks
with identical abundance of both nonhydroxy and 2hydroxy ceramides. Thus, this NL ESI mass spectrum
can be used to quantitate both nonhydroxy and 2-hydroxy
ceramide molecular species containing an 18-carbon
sphingosine backbone. Similarly, ESI tandem mass spectrometry with the NL of 242.2, 258.2, and 329.3 amu from
lipid extracts can be performed to identify and quantify
ceramide molecular species containing sphinganine (i.e.,
dihydrosphingosine) backbones. NL scanning of 268.2,
284.2, and 355.3 amu or other sets of NL spectra can be
used to analyze the sphingenine backbone with 20 carbon
homologs or other sphingosine analogs, respectively, in
the lipid extracts of biological samples. It should be
mentioned that many other approaches have recently been
developed to qualitatively and quantitatively analyze
ceramide molecular species under various conditions (Gu
et al., 1997; Mano et al., 1997; Raith & Neubert, 1998,
2000; Liebisch et al., 1999; Thomas et al., 1999; Sullards,
2000; Hsu & Turk, 2002). Analysis of ceramide molecular
species should specifically consider the risks and benefits
of each of these alternative methodologies that best fits
individual experimental goals.

mass spectrometry through corroborative analyses of head


groups and acyl moieties leading to definitive molecular
species identification including isobaric molecular species.
Specifically, NL scanning of 59.1, 183.1, or 189.1 amu
corresponding to trimethylamine, phosphocholine, or
lithiated cholinephosphate (or 205.1 amu for sodiated
cholinephosphate) can be used to identify the head group
of choline-containing phospholipids under the stated
conditions (Table 2; Fig. 12; Han & Gross, 1995; Hsu,
Bohrer, & Turk, 1998b). Mass spectra of extracts of murine
myocardium obtained from the NL scanning of 59.1 and
183.1 amu show lithiated molecular ions as well as low
abundant sodiated molecular ions (Fig. 12). Sodiated
PtdCho molecular species overlap with lithiated PlsCho
molecular species in the spectra. This complication can be
resolved by comparing these mass spectra with spectra
obtained from either the NL scanning of 189.1 amu
(selective for lithiated PC molecular ions) under the
identical conditions (Fig. 12) or the NL scanning of
50.0 amu (corresponding to the loss of chloromethane)
under the conditions for the analysis of anionic phospholipids (Fig. 12). After comparison of these NL mass spectra
(Fig. 12), it can be concluded that quantitation may also be
made (or prior results substantiated) from the mass spectra
of the NL 50.0 or 59.1 amu under conditions using an
optimal collisional activation energy (Fig. 12).
It should be pointed out that the differences of
aliphatic chain linkages to glycerol or to the sphingosine
backbone result in different fragment patterns between
PtdCho, PlsCho, and SM, which, in turn, affects the profiles
of choline-containing phospholipids after NL scanning of
59.1, 183.1, or 189.1 under different collision energies
employed (Fig. 12). However, these fragmentation differences may be exploited to identify individual molecular
species from each of these classes or subclasses in addition
to the distinction of SM molecular species from PC species
by using the nitrogen rule. The discrimination of PlsCho
and AlkCho molecular species can be made following the
treatment of a part of a lipid extract with acid vapor as
discussed in last subsection or by using tandem mass
spectrometry (Hsu et al., 2003).
Characterization of acyl chains in PC molecular
species, determination of the regiospecificity of the
aliphatic chains, and identification of molecular species
in the isobaric ion peaks can be made from their chlorine
adducts (see above) as previously described (Han & Gross,
1995). Alternatively, a much more accurate way to determine the regiospecificity of PC molecular species has
recently been reported using ion trap technology by Ekroos
et al. (2003). Characterization may also be made from the
NL analyses of all potentially-occurring fatty acids in PC
molecular species including 228.2 (14:0 FA), 254.2 (16:1),
256.2 (16:0 FA), 280.2 (18:2 FA), 282.2 (18:1 FA), 184.2
(18:0 FA), 304.3 (20:4 FA), 328.3 (22:6 FA), etc. with the

3. Analyses of Electrically Neutral but


Polar Lipid Molecular Species

The diluted lipid extract is left at alkaline pH as in last step.


The ESI mass spectrometer is switched to the positive-ion
mode to select for positive ions. Under these experimental
conditions, any lipid classes that contain a negative charge
(including anionic lipids and weak anionic lipids as
discussed in last two subsections) can not easily form
positively-charged ions during the ESI process and largely
remain at the positive potential end plate. This separation
of charged ions results in a de facto separation of lipid
classes in the ion source. Therefore, this separation greatly
reduces the effective lipid concentration during the
ionization process. The charge selection for anionic or
weak anionic lipids in negative-ion mode as discussed
above similarly yields a reduction of effective anionic or
weak anionic lipid concentration during the ionization
process. Thus, it greatly reduces the lipidlipid interactions/aggregation in the sprayed droplets and, in turn,
enhances the linear dynamic range of lipid quantitation.
Molecular species of choline-containing phospholipids
(e.g., PC, lysoPC, and SM), glycolipids, TAG, etc. are all
easily ionized as their small cation adducts (e.g., lithium
adducts) directly from the lipid extracts (Fig. 11; Han et al.,
1996, 2002; Hsu, Bohrer, & Turk, 1998b; Han & Gross,
2001; Hsu & Turk, 2001).
The pseudomolecular ions of choline-containing
phospholipids can be further characterized by tandem
16

SHOTGUN LIPIDOMICS BY ESI/MS

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A
FIGURE 12. Tandem ESI mass spectrometric analyses of choline glycerophospholipids directly from a

mouse myocardial lipid extract by neutral loss scanning of 50.0, 59.1, 183.1, or 189.1 in either negative- or
positive-ion mode. The lipid extract is identical to the one used for the analysis of anionic phospholipids
shown in Figure 8. The experimental conditions for neutral loss (NL) scanning were listed in Table 2 with a
collision gas pressure of 1 mTorr.

correction for 13C isotope effects (see Discussion in the


next section). Quantitation of PC and/or SM individual
molecular species in the diluted lipid extracts can be made
by comparing the intensity of each identified ion peak with
a selected internal standard (or standards) and appropriate
correction for 13C isotope effects (Han & Gross, 2001;
Han, 2002). It should be emphasized again that PlsCho

molecular species, like PlsEtn species, are acid-labile.


Thus, acidic conditions must be avoided for the analyses
of biological samples containing vinyl ether subclasses.
It should also be noted that the amide proton in SM
molecular species is a weak acid (although very weak), the
electrical characteristics of the solvent (i.e., the presence of
LiOH) tends to polarize the bond to the amide nitrogen,
17

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HAN AND GROSS

Author Proof

which facilitates its reduction in an electric field and


increases the generation of anionic species. Therefore, this
tendency results in a relatively lower ionization efficiency
of SM species in comparison to PC species in the presence
of base and an underestimation of SM mass content in the
lipid extract if too much LiOH is added. Therefore, a SM
analog should be used as an internal standard for quantitation of SM molecular species when accurate quantification is necessary. Alternatively, quantitation of SM species
from NL scanning of 50 amu from the lipid extract without
the addition of LiOH can be performed to verify that the
correct amount of LiOH is present and to refine the
accuracy of SM mass quantitation.
Glyco(sphingo)lipids such as cerebrosides can also be
analyzed as their lithiated adducts under the experimental
conditions employed (i.e., in the presence of LiOH and in
positive-ion mode) and can be quantitated by comparing
the intensity of each identified ion peak with a selected
internal standard (e.g., perdeuterated palmitoyl cerebroside) and appropriate correction for 13C isotope effects
(Han et al., 2002). The lithium adducts of this class of lipids
as well as other more complicated glyco(sphingo)lipids
have been extensively characterized by Hsu & Turk (2001).
These observed fragment patterns can be used to identify
ion peaks corresponding to the glyco(sphingo)lipid molecular species directly from a lipid extract (Table 2).
Similarly, lysoPC molecular species can be identified as
PC molecular species with NL scanning of 59.1, 183.1, or
189.1 amu and quantitated by comparison of the intensity
of each identified ion peak with a selected internal standard
(e.g., 17:0 lysoPtdCho) and appropriate correction for
13
C isotope effects (Mancuso et al., 2003). Alternatively,
NL scanning of 50.0 amu (corresponding to the loss of
chloromethane) under the conditions without the presence
of LiOH can be used to identify and quantify lysoPC
molecular species similar to methods used for their parent
PC molecular species as discussed in the last paragraph. PI
scanning of m/z 184.1 can also be used to identify and
quantify lysoPC molecular species directly from lipid
extracts in the presence of ammonium acetate as previously
described (Liebisch et al., 2002). The regioisomers of these
lysophospholipids can be readily discriminated as sodium
or lithium adducts directly from lipid extracts by comparisons of their fragmentation patterns (Han & Gross,
1996; Khaselev & Murphy, 2000; Hsu et al., 2003).
As discussed in section III, although TAGs that typically contain three long acyl chains in biological samples
are normally categorized as non-polar lipids, ESI/MS of
lipid extracts show very abundant small cation adducts
of TAGs such as their ammonium, lithium, or sodium
counterparts (Duffin, Henion, & Shieh, 1991; Hsu & Turk,
1999; Han et al., 2000; Han & Gross, 2001). However,
because of the absence of a dominant dipole potential in
TAG molecules, each double bond or each methylene

segment substantially affects to the molecular dipole


potential. Thus, the ionization efficiency of TAG molecular
species greatly depends on the acyl chain moieties. We
have determined the ionization efficiencies (i.e., sensitivity
correction factors) of 23 different TAG molecular species
varying from 42 to 60 of total carbon numbers and from 0 to
12 of total double bonds in three acyl chains relative to the
selected internal standard (i.e., T17:1 TAG) (Han & Gross,
2001). Through a least-square regressive nonlinear curve
fitting of these sensitivity correction factors, an algorithm
has been developed as follows:

18

y 4:4979 0:3441p 0:1269q 4:845


 103 p  q 9:9  104 q2

where y is a correction factor for sensitivity effect relative


to T17:1 TAG, q is the total carbon number in the three acyl
chains of a TAG species, and p is the number of total double
bonds in a TAG species (Han & Gross, 2001).
Even with the help of this algorithm, quantitation of
TAG molecular species directly from a lipid extract of a
biological sample is still complex and the presence of
multiple complicate isobaric ion peaks and severe overlaps
between TAG and PC molecular species further compound
analysis. To overcome these difficulties, we have developed a 2D mass spectrometric technique based on NL
scanning of all naturally-occurring fatty acids to fingerprint
most, if not all, of TAG molecular species in the lipid
extract (Fig. 13) and to quantitate these identified species in
combination with the sensitivity correction factors and
correction for 13C isotope effects (Han & Gross, 2001).
Therefore, fingerprinting and quantitation of individual
TAG molecular species directly from chloroform extracts
of biological samples can be achieved with an error of
approximately 10%, which has been routinely attained in
our laboratories (Han & Gross, 2001; Finck et al., 2002,
2003; Listenberger et al., 2003; Mancuso et al., 2003;
Newberry et al., 2003). A similar strategy may be applied to
the fingerprinting and quantitation of DAG molecular
species. The regiospecificity of DAG molecular species
may be determined by a method similar to the analysis of
neutral ether lipids as described by Kuksis and colleagues
(Hartvigsen et al., 2001).
4. Analyses of Specialized Lipids
and Lipid Metabolites

Acylcarnitines in the lipid extracts can be readily analyzed


by PI scanning of m/z 85 corresponding to a carnitine
derivative (Table 2), which has been extensively used for
biological studies and clinical diagnoses (Rashed et al.,
1994; Ford et al., 1996; Johnson, Mills, & Clayton, 1996;
Vreken et al., 1999; McClellan, Quarmby, & Yost, 2002;
Moeder et al., 2003). We generally assess the content of

SHOTGUN LIPIDOMICS BY ESI/MS

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A
FIGURE 13. A two-dimensional ESI mass spectrometric fingerprint and quantitation of triacylglycerol

molecular species of a mouse myocardial lipid extract by neutral loss scanning in positive-ion mode in the
presence of LiOH. All neutral loss (NL) scans displayed are normalized to the base peak in the individual
scan.

cholesterol and its esters in lipid extracts by employing


fluorometric method (Han et al., 2002, 2003a). However,
Sandhoff et al. (1999) have developed a method to
quantitate cholesterol and its derivatives by ESI tandem
mass spectrometry after a simple one-step chemical
derivatization of cholesterol to cholesterol-3-sulfate by a
sulfur trioxide-pyridine complex. Duffin and colleagues
(Duffin et al., 2000) were able to profile individual
molecular species of cholesterol esters by PI scanning of
m/z 369.3 (corresponding to a sterol derivative) from

ammoniated molecular ions (Table 2). Very recently,


Seal et al. (2003) have reported a silver ion-coordinated
ionspray mass spectrometric method to analyze cholesterol
ester molecular species. These and other specialized
methods continue to be developed at rapid rates. Direct
analysis of sphingolipid metabolites from lipid extracts
still needs to be developed. However, Lieser et al. (2003)
have recently reported an approach for the quantitative
analysis of sphingosine and sphinganine by using HPLCESI/MS/MS.
19

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B. Technical Aspects Facilitating the Successful


Performance of ESI/MS Employing Intrasource
Separation of Lipid Classes

Author Proof

added for the analyses of weak anionic lipid molecular


species as described above. The advantage of using the
NH4Cl/NH4OH paired system is the longevity of the ion
source between cleanings as well as the ease of maintenance for the entire instrument. However, many features
that are unique to lithium adducts are lost with ammonium
adduct and thus, some classes of lipids can not be quantitated (e.g., TAG) or extensively analyzed (e.g., PC and
glycolipids) directly from a crude lipid extract of biological sample with the NH4Cl/NH4OH system. For
example, protonated PC molecular ions are predominant
in the presence of NH4Cl/NH4OH. Therefore, m/z 184.1 is
the only abundant product ion from a protonated PC
molecular ion. However, in the case of using LiCl/LiOH
system, the head group of PC molecular species can be
characterized by NL of either 59.1 or 183.1 amu and in
addition, the acyl constituents of PC molecular species can
be determined by NL scanning of all naturally occurring
fatty acids.

1. Lipid Extraction

Lipid extraction is one of the key steps to the successful


analyses of cellular lipidomes by ESI/MS in general and
by intrasource separation technique of ESI/MS in particular. Commonly, lipid samples from biological sources
are extracted using a mixture of chloroform and methanol
based on the Folch method (Folch, Lees, & Stanley,
1957), or the modified method of Bligh & Dyer (1959).
Regardless how carefully you conduct your extractions,
small amount of water and ions are carried into the
chloroform extracts. Therefore, if a high salt concentration present in aqueous phase during the chloroform
extraction, even small residual contamination of the chloroform extract with its aqueous phase constituents will result
in a high chemical noise level because of the presence
of high concentrations of lower molecular weight counterions and other compounds. Therefore, it is recommended that an additional Bligh and Dyer extraction
against an aqueous phase with a lower salt concentration
be used to clean up the lipid extracts in the case of
samples that are contaminated with sample counterions.
Although acidic conditions of the aqueous phase can
improve the extraction efficiency for acidic lipids such as
PtdH, acyl CoA, acyl carnitine, etc., plasmalogen molecular species are very labile under acidic conditions
(Ford, Rosenbloom, & Gross, 1992; Kayganich & Murphy,
1992). Detergents complicate the analyses of lipid molecular species by ESI/MS and thus, detergents should be
avoided when possible. If present, great care needs to
be exercised in removing as much of the added detergents
as possible.
A low concentration LiCl solution (50 mM in aqueous
phase) during lipid extraction works best in our experience.
This weakly acidic solution results in the best selectivity in
negative-ion ESI/MS for anionic lipid analyses from PE
species since anionic lipids are present in net negatively
charged form, whereas PE lipids existed as zwitterions
under the conditions employed. Therefore, ESI/MS in
negative-ion mode showed substantially different ionization efficiency for these two groups of lipids, resulting in
the selective ionization and effective separation of these
two-groups of lipids in the ion source without chromatography. In addition, the extraction efficiency of acidic
lipids can be improved by using this weakly acidic solution
and the degradation of plasmalogen molecular species
(typically manifest at pH 4 or less) does not occur under
these conditions.
Alternatively, an NH4Cl solution can be used to
replace LiCl for the same purpose. Thus, NH4OH could be
20

2. Lipid Concentration

Many studies have demonstrated that ESI of lipids is a


concentration-dependent process (e.g., Han & Gross,
1994; Delong et al., 2001; Koivusalo et al., 2001). At
concentration higher than 0.1 nmol/mL (i.e., 0.1 mM), the
effects of acyl chain length and unsaturation on ionization
efficiency are apparent (Koivusalo et al., 2001; Zacarias,
Bolanowski, & Bhatnagar, 2002). However, at low concentration of lipids, a linear correlation of ion intensity with
the lipid concentration of each class is present (Han &
Gross, 1994; Kim, Wang, & Ma, 1994; Delong et al., 2001;
Koivusalo et al., 2001).
Alterations in signal intensity at high concentration
are largely because of the lipidlipid interactions and/or
aggregation. Specifically, at the low concentration, lipid
lipid interactions are rare and ionization efficiency of lipid
mixtures is only dependent on the electrical propensity of
each lipid molecular species to lose or capture a charge,
which is predominantly determined by the nature of polar
head groups. Therefore, an approximately linear dependence of ion abundance on the lipid concentration of each
class in a range of amol/mL to pmol/mL can be obtained.
Since lipid packing (or aggregation) is highly dependent on
the physical properties of lipids (Han & Gross, 1990,
1991), when the concentration of lipids in the infusion
solution increases to the point where lipidlipid interactions become apparent, the linear relationship between ion
intensity and lipid concentration is no longer true. Thus,
the ionization efficiency is also dependent on acyl chain
length and unsaturation of lipids. Therefore, if the sensitivity of an instrument permits, it is always favorable to
quantitate lipids at a low concentration of lipid in solution
(e.g., <10 pmol/mL).
10 uM

SHOTGUN LIPIDOMICS BY ESI/MS

3. Internal Standards

PI internal std

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Author Proof

For quantitation of lipid molecular species in a biological


sample, or comparison of lipid profiles between lipid
extracts of biological samples, appropriate amounts of
suitable internal standards have to be added to each homogenate prior to lipid extraction. In most cases, lipid content
is normalized to the tissue or cell protein content. However,
tissue wet or dry weight, cell number, and DNA content are
also frequently used by other investigators in many cases.
Each normalization has benefits and detriments depending on the physiologic or pathologic system studied. Lipid
molecular species we typically utilize include 15:0-15:0
PtdGro, 15:0-15:0 PtdSer, 15:0-15:0 PtdEtn, 14:1-14:1
PtdCho, 17:0 lysoPtdCho, 2H35-N18:0 GalC, N16:0
sulfatide, N17:0 ceramide, T17:1 TAG, 2H31-16:0 NEFA,
and 12:0 acylcarnitine as internal standards for most of
the biological samples. These internal standards can be
selected because they generally only represent 1% of
endogenous cellular lipid mass for most of cell types.
However, a survey for the absence of any isobaric peaks
from the endogenous cellular lipids coexisting with the
pseudomolecular ions of these molecular species has to
be performed prior to the usage of these lipids as internal
standards. It would be beneficial to include internal
standards for cardiolipin, PtdH, and SM for accurate
quantitation of these classes as discussed above.
Depending on the interests in the analyses of lipid
classes and the source of the samples, discretion on the
appropriate utilization of internal standards is always
advised. For example, 2H35-N18:0 GalC and N16:0 sulfatide are mainly used for the analyses of cerebrosides and
sulfatides in brain samples. The internal standard 15:015:0 PtdGro is for quantitation of anionic phospholipids
including cardiolipin, PtdH, PtdGro, and PtdIns; 14:1-14:1
PtdCho is for PC and SM; 15:0-15:0 PtdEtn is for PE and
lysoPE; and other internal standards stated above are
for their own classes. The amounts of internal standards
necessary for lipid quantitation may be varied for different
kinds of samples and it is suggested that identification of
separated samples and concentrations of internal standard
in pilot experiments. However, following concentrations
(nmol/mg of protein) can normally be used as a start point:
15:0-15:0 PtdGro, 2; 15:0-15:0 PtdSer, 1; 15:0-15:0
PtdEtn, 15; 14:1-14:1 PtdCho, 15; 17:0 lysoPtdCho, 1.5;
2
H35-N18:0 GalC, 20; N16:0 sulfatide, 10; N17:0
ceramide, 0.05; T17:1 TAG, 5; 2H31-16:0 NEFA, 2; or
12:0 acylcarnitine, 0.3.

ferent laboratories, the importance of applicable standard


instrument parameters for data acquisition need to be
discussed. Specifically, accurate quantitation of cellular
lipidomes depends on utilization of appropriate collision
energies, mass calibration, and ionization parameters.
First, appropriate caution in employing ESI tandem mass
spectrometry for quantitation of individual molecular
species of each class of lipids must be exercised, because
the fragmentation patterns of each lipid molecular species
depend on both the applied energy for collision-induced
dissociation and the structure of individual molecular
species (Han & Gross, 1995, 2001; Sullards, 2000; Delong
et al., 2001; Han, 2002). Changes in applied collision
energy alter the kinetics of individual fragmentation ???!
pathways resulting in changes in the distribution of the
observed fragment ions. In some cases differential energydependent fragmentation can lead to substantial errors in
mass analysis as previously been documented for TAG
species because of aliphatic chain differences (Han &
Gross, 2001) or PC and SM because of different backbone
covalent structures, which fragment differently at different collision energies (Han & Gross, 1995). Similarly,
aliphatic chain differences in naturally occurring phospholipids (e.g., 16:0-18:1 PtdEtn to 16:0-22:6 PtdEtn) can
lead to approximately 2-fold differences in the intensity of NO!
Maybe
the sn-2 acyl carbonium ion after ESI depending on the
number of carbon atoms, olefinic linkages and activation
energies employed in the negative ion mode (Han & Gross,
1995). Thus, it is important to closely control fragmentation energies and to both utilize appropriate internal
standards for the lipid classes and molecular species as well
as ratiometrically quantify each individual species so that
identical physical parameters are compared. Profiling by
NL and/or PI scanning should always be performed and
justified with appropriate standards (see later for further
discussion) and careful attention to collision-induced
activation energy must be exercised if accurate quantitation is needed.
For example, Figure 14 shows the effects of collision
energies on the alterations in pseudomolecular ion intensities of an equimolar mixture of ceramide molecular
species. When a relatively low collision energy is used
(e.g., <30 eV), not only does the efficiency of collisional
dissociation diminish, but also a shift in the abundance of
ions toward low molecular weight/unsaturated species is
observed (Fig. 14A). In contrast, when a relatively higher
collision energy is employed (e.g., >35 eV), the highmolecular-weight ceramide species are more abundant
in comparison to low-molecular-weight species in NL
mass spectra of ceramides (Fig. 14B). The reason underlying this phenomena likely involves the differential
change of entropy and enthalpy of the compounds (Han,
2002). Delong et al. (2001) conducted an extensive
investigation on the effects of collision energies on the

4. Instrument Parameters for Shotgun


ESI/MS of Lipids

Although there are no universal instrument conditions


broadly applicable for different instruments used in dif-

21

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Author Proof

A
FIGURE 14. Neutral loss tandem mass spectra of an equimolar mixture of ceramides under different

collision energies employed. The solution of equimolar ceramide mixture (10 nM each) in chloroform/
methanol (1:1, by volume) was directly infused into the ESI ion source at a flow rate 1 mL/min in the presence
of LiOH. ESI tandem mass spectra with the neutral loss of 240.2 of the ceramide mixture under collisional
activation energy of 28 eV (A) or 36 eV (B) were acquired. (Reprinted from Han (2002) with permission
Elsevier Science (USA), Copyright 2002.)

relative intensities of different molecular species in


different classes. Figure 15 shows the summary of results
from their study, suggesting again the dramatic effects of
collision energy and molecular structure on the quantita22

tion. We point out that these factors notwithstanding,


ratiometric comparisons are still quite useful because the
relative fragmentation rates of identical molecular species
reflect the intrinsic chemical properties of the species in

SHOTGUN LIPIDOMICS BY ESI/MS

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Author Proof

A
FIGURE 15. Differential effect of collisional activation energy on head group fragmentation efficiency.

Equimolar mixtures of 14:0-14:0 PtdCho (m/z 678) and 16:0-18:1 PtdCho (m/z 760) (A), 16:0-16:0 PtdEtn
(m/z 692) and 16:0-22:6 PtdEtn (m/z 792) (B), and 14:0-14:0 PtdSer (m/z 680) and 16:0-18:1 PtdSer (m/z
762) (C) were analyzed using PI scanning at collisional activation energies ranging from 15 to 40 eV. Plots
A, B, and C show the increase in slope as collisional energy increases. The slopes at different collision
energies were derived by plotting each pair of phospholipids relative to the peak area of the low molecular
mass species. The peak area ratios of the higher molecular mass species to the lower molecular mass species
in each pair are plotted in (D) as a function of collision energy to determine the collision energy at a ratio of 1.
(Reprinted from Delong et al., 2001 with permission from The American Society for Biochemistry and
Molecular Biology, Inc., Copyright 2001.)

dilute solution. Ratiometric comparison of molecular


species from a given class in different states, such as
health and disease, can provide important clues to molecular mechanisms underlying the disease process under
study.
The tuning and calibration of a mass spectrometer may
greatly influence the quantitative analyses of lipids when
using tandem mass spectrometry, particularly in NL mode.
The mass accuracy of the spectrometry during PI or NL
scanning depends not only upon the mass accuracy of both
the first and second mass analyzers, but also upon the mass
difference between these two analyzers. In other words,

during the NL and PI scannings, the quadrupoles must be


correctly calibrated to transmit the desired ions at the
appropriate time. Another issue comes from approximating either the lost mass in the NL scanning mode or the
selected ion in the PI scanning mode. For example, such
artifacts can result from setting the NL of stearic acid at the
approximation of 284 amu instead of 284.3 amu or setting
the PI of stearate at the approximation of m/z 283 instead
of m/z 283.3. It was demonstrated that when the NL mass
intently offset by as little as 0.4 amu, there was an error of
over 50% in quantitation as well as a dramatic reduction in
overall detection sensitivity (Fig. 16).
23

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A
FIGURE 16. Neutral loss tandem mass spectra of an equimolar mixture of ceramides under different

neutral loss offsets. The solution of equimolar ceramide mixture (10 nM each) in chloroform/methanol
(1:1, by volume) was directly infused into the ESI ion source at a flow rate 1 mL/min in the presence of LiOH.
ESI tandem mass spectra with the neutral loss of 256.2 (A) and 255.8 (B) of the ceramide mixture under a
collisional activation energy of 32 eV were acquired. The horizontal bars in panel A indicate the individual
peak intensity after the correction for 13C isotope effects.

24

SHOTGUN LIPIDOMICS BY ESI/MS

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Author Proof

!!!

The mass resolution of the ion peaks in PI mass spectra


is much lower than those observed in either product-ion
spectra or in NL spectra in most cases. We have found that
this peak broadening depends in part on the collision gas
pressure. The higher the collision gas pressure, the lower
the peak resolution. This effect is likely because of a timelag resulting from a different free path experienced by the
interrogated ions when the collision gas pressure increases.
Although further studies are needed to prove this hypothesis, a reduction of the collision gas pressure to certain
degree is always beneficial if the peak broadening is an
issue in the PI analyses of lipids and other molecules.

specific class of lipids using a calibration curve. Relative


abundance of molecular species of a specific lipid class
were used to compare lipid alterations induced by various
stimuli (Delong et al., 2001). Recently, Shevchenko and
colleagues (Ekroos et al., 2002a; Ekroos & Shevchenko,
2002b) have developed an approach to measure the relative
changes of major classes of phospholipids using a mixture
of isotopically-labeled endogenous lipids as a comprehensive internal standards. It has been observed that
the ionization efficiency of a class of phospholipids is more
dependent on the chemistry of the head group than on
the fatty acid moieties within a phospholipid class and the
relationship between ion counts and lipid amounts is linear
in certain range of concentration (Han & Gross, 1994;
Kim, Wang, & Ma, 1994). Based on these observations,
investigators (Fridriksson et al., 1999; Ivanova et al., 2001)
first determined relative ionization sensitivities in the
positive- and negative-ion modes. Using these relative
sensitivity factors, the relative composition of the total cell
lipid extract or subcellular fractions was determined.
In a very early study, we showed that the ESI efficiency
of lipid molecular species are predominantly dependent on
the lipid polar head group and only modestly dependent
on the physical properties of acyl chains in the concentration range of pmol/mL or lower (Han & Gross, 1994). Two
reasons likely underlie this observation. First, ESI of lipids
under conditions typically employed (e.g., a low lipid
concentration in the infusion solution where lipidlipid
interactions are diminutive) is dominated by the electrical
propensities of the lipid polar head groups as discussed
above. Second, the stability of generated ions is rarely
dependent on acyl chain properties such as chain length
and unsaturation index. In the same study, we also demonstrated a linear relationship between the ion counts and
the amounts of lipid consumed in a low concentration
regime (Han & Gross, 1994). This linear relationship was
independently demonstrated by multiple other investigators (e.g., Kim, Wang, & Ma, 1994; Lehmann et al., 1997;
Delong et al., 2001; Koivusalo et al., 2001) and serves as
benchmark for quantitative ESI/MS analysis when accuracy 5% is desired. As an extreme case, we examined the
relationship between palmitoyl lysoPtdCho and dipalmitoyl PtdCho. Addition of various amounts of palmitoyl
lysoPtdCho to a solution of containing dipalmitoyl PtdCho
resulted in a linear correlation between their molar ratio
and the ratio of their respective sodiated molecular ion
peak intensities in positive-ion ESI mass spectra after
correction for 13C isotope effects with a slope of 1.00 and a
correlation coefficient factor (g2) of 0.998 (Han & Gross,
1994). Similar results have been observed with other
systems. As the lipid concentration of the infusion solution
increases, lipidlipid interactions become severe and the
linear relationship between ion intensity and lipid concentration is no longer observed as demonstrated by

V. QUANTITATION OF CELLULAR
LIPIDOMES BY ESI/MS

Ideally, quantitation of any compound by mass spectrometry only can be accurately made by comparison of its
peak intensity with that of a stable-isotope incorporated
and chemically-identical internal standard analyzed under
identical experimental conditions. Such kinds of requirements can be achieved for the quantitation of a few known
lipids. For example, Murphy and colleagues (Harrison,
Clay, & Murphy, 1999), used a stable-isotope incorporated
internal standard to measure one particular component of
the mixture. However, it is infeasible to use hundreds to
thousands of internal standards for the quantitative analyses of a complex lipid mixture (e.g., a crude lipid extract
of a biological sample) in which thousands or even tens of
thousands of lipid molecular species are present. Literally,
therefore, many different approaches (see below) have
been employed to quantitate or semi-quantitate lipid
molecular species or a class of lipids.
In early experiments, we employed representative
internal standards to quantify major lipid classes in platelets (Han et al., 1996). Similarly, by addition of unnatural
phospholipid(s) that possess m/z outside of the molecular
weight envelope of the naturally occurring molecular
species, Lehmann et al. (1997) assessed the rat bile PtdCho
molecular species and Liebisch et al. (2002) quantified
lysoPtdCho molecular species in plasma samples by ESI
tandem mass spectrometry. Multiple internal standards that
are distributed through the expected molecular weight
region were demonstrated by Brugger et al. (1997) in
analyses of PC molecular species by PI scanning of m/z
184. This approach has also been employed by Welti et al.
(2002, 2003) for the quantitation of multiple lipid classes in
plants. Blom et al. (2001) also employed multiple internal
standards for each class of lipids across the molecular
weight region of interest to analyze the class of lipids
including PC, PE, PtdSer, and SM.
Some investigators (e.g., Liebisch et al., 1999;
Zacarias, Bolanowski, & Bhatnagar, 2002) quantitated a

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multiple investigators (Delong et al., 2001; Koivusalo


et al., 2001; Zacarias, Bolanowski, & Bhatnagar, 2002).
We point out that this nonlinear relationship is mainly
because of the lipid-lipid interactions and is different from
the generally called ion suppression.
Accordingly, we believe that direct quantitation of a
class of lipid molecular species in lipid extracts can be
made by comparisons of the individual molecular ion peak
intensity with an internal standard for each polar lipid class
after correction for 13C isotope effects within requisite
biological experimental error (i.e., approximately 5%)
(Han & Gross, 1994; Han et al., 1996, 2002). However,
when the polarity of the head group in a lipid class is weak
(e.g., TAG), the effects of acyl chain physical properties on
ionization and ion stability become apparent and need to be
considered. Thus, an algorithm collecting for the sensitivity of each individual molecular species relative to the
selected internal standard needs to be developed and
executed in future analyses of multiple lipidomes (see
Eq. [1]; Han & Gross, 2001).
The necessity for the correction for 13C isotope effects
in lipid quantitation is derived from basic chemical
principles. Two main types of 13C isotope effects need to
be considered during quantitative lipid analysis in generating cellular lipidomes as we have described previously
(Han & Gross, 2001). The first effect comes from the
carbon number difference between a given molecular
species and the selected internal standard and can be
calculated by multiplying the monoisotopic peak intensity
by a calculated factor as follows:

peaks. Because m  1 in almost all cases, Eq. [3] can be


simplified into
Z2  1 6:05  105 m2 IM2 =IM

Z1 1 0:011n 0:0112 nn 1=2=


1 0:011s 0:0112 ss 1=2

where Z1 is a type I 13C isotope correction factor, n is the


total carbon number in the molecular species of interest,
and s is the total carbon number of the selected internal
standard. The level of this type of correction is <10% in
most cases.
The second 13C isotope effect is because of the
overlapping nature of the pseudomolecular ion peak of the
species of interest with the M 2 isotope peak from
another species that has a 2-Da lower mass (e.g., the 13C
isotope effect of 16:0-18:2 PtdCho on 16:0-18:1 PtdCho).
This effect can be corrected by multiplying the monoisotopic peak intensity by a second factor as follows:
Z2 1 IM2 =IM 0:0112 mm 1=2

where Z2 is a type II 13C isotope correction factor, m is the


total carbon number in the molecular species with lower
molecular mass, and IM2 and IM are peak intensities of
ions at m/z of (M2) and M, respectively. This effect is
random and the degree of this type of correction is
dependent upon the relative intensities of the involved ion
26

It should be noted that the Li isotope represents 7.4%


of the natural abundance of 7Li. However, the intensity of
this isotope ion peak is proportional to the 7Li ion peak
intensity in positive-ion ESI/MS for all molecular species
including the selected internal standard. Therefore, this
isotope effect does not to be considered during direct ion
peak comparisons. The secondary effects of 6Li isotope to
the 13C isotope effect that results from the carbon number
difference is very small and can usually be neglected.
Only in the cases where SM species and PC species are
adjacent such as N18:0 SM (m/z 737.6) and 16:0-16:1
PtdCho (m/z 738.6) does this isotope effect of PtdCho on
SM ion abundance need to be considered. In the latter case,
we have previously used to our advantage the differential fragmentation rates of PCs and SMs to discriminate
contributions from each class if additional clarification is
necessary. Perform mild AH for SM analysis
VI. COMPARISONS BETWEEN DIFFERENT ESI/MS
APPROACHES TO THE GLOBAL QUANTITATIVE
ANALYSES OF CELLULAR LIPIDOMES

Since the initial use of ESI/MS in the characterization of


DAGs by Duffin, Henion, & Shieh (1991) and of platelet
activating factor by Weintraub, Pinckard, & Hail (1991),
multiple ESI/MS techniques have been developed and
numerous applications for the analyses of various classes,
subclasses, and/or molecular species of lipids from different sources have been reported (see recent reviews for
references: Griffiths, 2003; Han & Gross, 2003; Pulfer &
Murphy, 2003; Welti & Wang, 2004). These studies have
demonstrated the unparalleled sensitivity, specificity, and
efficiency of ESI/MS for the analysis of lipids. Through
appropriate sample preparation, instrument setting, and/
or experimental design, ESI mass spectrometry including
tandem mass spectrometry allows: (1) the rapid and
efficient analysis of lipid classes, subclasses, and individual molecular species directly from crude extracts; (2) a
dramatically higher signal to noise ratio in comparison to
other mass spectrometric approaches such as FAB/MS and
MALDI/TOF-MS; (3) a nearly linear relationship between
the relative intensities of pseudomolecular ions and the
mass of individual lipids over a 10,000-fold dynamic range
in the low concentration regime; (4) dependence of ion
intensity on the nature of polar head group but rarely on
acyl chain physical properties such as chain length and
unsaturation index; and (5) excellent reproducibility of
measurements from an identical sample (<5% of experimental error).

SHOTGUN LIPIDOMICS BY ESI/MS

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Although it has been shown that there are many advantages of ESI/MS for lipid analysis over other analytical
methods and many techniques have also been developed
(see Griffiths, 2003; Han & Gross, 2003; Pulfer & Murphy,
2003; Welti & Wang, 2004 for recent reviews), the
applications of ESI/MS in the quantitative analyses of
global cellular lipidomes are still limited. Most of the
previous studies on lipidomes by ESI/MS were mainly
focused on the characterization of lipids (see recent
reviews for references: Griffiths, 2003; Pulfer & Murphy,
2003). Furthermore, compared to the dramatic progress
in genomics and proteomics, progress in lipidomics, an
important branch of metaboliomics, still lags because of
the complicated nature of cellular lipidomes and because
less attention has been paid on lipidomic studies than on
genomic and proteomic research.
There are four main different strategies currently
employed for the global analyses of cellular lipidomes
using ESI/MS. These approaches include methods employing HPLC-ESI/MS, ESI tandem mass spectrometry,
FTMS, and the intrasource separation ESI/MS approach.
Each approach has specific advantages over other approaches but as is typically the case in analytical science,
there are strengths and weaknesses inherent in each and
appropriate strategies that integrate the strengths of each
approach to the goal of the specific study are advised.
We specifically point out that these approaches are not
independent but they are inter-related. Further development of each approach is still needed for global analysis of
cellular lipidomes to molecular detail. Which approach is
best suited for a particular application is largely dependent
on the requirements of the study (e.g., the available sample
size, the requisite accuracy of the analytical results, etc.)
and the available instruments. The remainder of this
section will discuss each approach in some detail.

molecular species of each lipid class by using one related


internal standard of the class after correction for 13C
isotope effects within experimental error (i.e., 5%).
Furthermore, for the first time, we were able to identify
multiple PC molecular species in positive-ion mode and PE
molecular species in negative-ion mode from a crude lipid
extract of a biological sample although the mass spectra
using those conditions were unrefined (Fig. 17). Currently
technical and instrumental advances and refinements have
led to dramatic increases in S/N with a resultant increased
depth of analytical penetration (compare Fig. 17 with
Figs. 9 and 11). The principles of intrasource separation
were first applied to analyze the lipidome in activated
platelets after thrombin stimulation to identify lipid alterations in activated platelets (the most dramatic example
of ligand-stimulated hydrolysis we know of in nature)
(Han et al., 1996). Alterations in the molecular structure
and regiospecificity of each lipid constituent were determined directly from lipid extracts of activated platelets
(Han & Gross, 1995, 1996).
There are multiple inherent advantages present in the
intrasource separation approach. The first is simplicity.
One can use appropriately selected internal standard to
quantitate all individual molecular species in each chargerelated lipid class or classes and can utilize selective
ionization propensities for the separation of lipid classes in
the ion source in the approach. The second advantage of the
approach is its high sensitivity. Global analyses of cellular
lipidomes from a lipid solution at a concentration less than
1 pmol/mL with a volume equal to or less than 100 mL can
be readily achieved by using this approach. The third
advantage of this approach is its relatively high accuracy
(i.e., errors 5%) for biological studies for most of
individual molecular species and lipid classes. The fourth
advantage is its broad application since more lipid classes
can be quantitatively analyzed using this approach than
other approaches to date. The fifth important characteristic
of this approach is its efficiency. A total lipid analysis from
a crude lipid extract can be readily achieved in less than
30 min. In addition, as discussed above, intrasource separation can lead to the reduction of lipidlipid interaction/
aggregation and, in turn, enhance the linear dynamic range
of lipid quantitation in this approach relative to many other
approaches.
There are two major weaknesses to direct lipidome
analysis using this approach. The first one is the contamination of the instrument with lithium ions. In our
experience, it becomes necessary to clean the ion source
every 68 weeks and tune the instrument frequently.
However, one can overcome this problem by replacing
LiClLiOH with NH4ClNH4OH during sample preparation, unless analyses (e.g., TAG analysis) that require
lithium are being performed. The second issue is that ion
suppression for low abundance ions must be considered.

A. Quantitation of Cellular Lipidome by the


Intrasource Separation Approach of ESI/MS

The principles of the intrasource separation approach were


established in a very early study (Han & Gross, 1994).
These principles include the demonstration of: (1) the
linearity between molecular ion intensity and lipid concentration in the infused solution, (2) the nearly identical
ionization efficiency of phospholipid molecular species
within each different lipid class and independence on the
physical properties of acyl chains in the concentration
range of pmol/mL or less, and (3) the use of charge specific
internal standards reflecting the electrical propensity for
different charged classes of phospholipids in both positiveand negative-ion mode. In addition, a very important and
inherent correction factor, i.e., 13C isotope effects, was also
recognized and employed in that study. Through this
approach, we were able to quantitate individual lipid

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A
FIGURE 17. The first ESI mass spectra of cellular phospholipids directly from a crude extract of human

erythrocyte plasma membrane. The positive-ion ESI mass spectrum (Panel A) displayed predominant PC
and SM molecular species, whereas the negative-ion ESI mass spectrum (Panel B) displayed abundant PE
molecular species in the presence of NaOH. (Reprinted from Han & Gross (1994) with permission from the
National Academy of Sciences, Copyright 1994.)

Generally, if the abundance of ions is lower than 3% of


the base peak intensity in a mass spectrum in the intrasource separation approach, these ion peaks are either
buried in the baseline or quantitation of low abundance
peaks is difficult by this approach alone. The total mass
of a class of lipids will not be significantly affected by
ignoring these low abundance species although important
information in them will be lost if these peaks are important
in an investigation. This problem can be solved with the
help of tandem mass spectrometry through which noisy
baselines can be eliminated and the amounts of lipids
corresponding to the low abundant ion peaks can be
28

estimated. Although tandem mass spectrometry was


mainly used to identify the molecular structure of individual species, to resolve the molecular species in
isobaric peaks, and to determine the regiospecificity of
each individual molecular species in conjunction with the
intrasource separation approach, tandem mass spectrometry was also used to refine the quantitation accuracy of
the mass content of low abundance molecular species
present in the sample in our previous studies. Alternatively,
HPLC methods we and others have developed for lipid
analyses can be employed (e.g., Gross & Sobel, 1980;
Creer & Gross, 1985).

SHOTGUN LIPIDOMICS BY ESI/MS

B. Quantitation of Cellular Lipidome by


Tandem Mass Spectrometry

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(see above). However, there are multiple issues that need


also to be considered. First, like the intrasource separation
approach, the concentration of lipid infusion solution has
to be low enough so that lipidlipid interactions will not
alter lipid profiles demonstrated by ESI tandem mass
spectrometry. Second, collision activation energy is a
parameter inherent in the specific manufacturers design
and instrumental model, or even within a given individual
mass spectrometer. Accordingly, calibration needs to be
carefully examined by individual investigators to provide
the appropriate balance of high sensitivity for detection of
lipids and minimize effects on differential fragmentation
in the alterations in a lipid profile. Third, the selected
internal standards should not only consider the coverage of
the range of interested lipid molecular species, but also
represent the diversity of lipid molecular species because
the unsaturation index is a major factor, which affects lipid
fragmentation kinetics after ESI (e.g., Han & Gross, 1995).
Finally, although the correction of type I 13C isotope effect
(see above) can be covered by a calibration curve that is
derived from the selected multiple internal standards,
the correction of type II 13C isotope effect still need to be
considered because this effect is random and can lead to
very large errors.

Tandem mass spectrometry including both PI scanning


and NL scanning is a very powerful technique for lipid
analysis (e.g., Han & Gross, 2003). In PI scanning, a unique
fragment ion from a class or a subgroup of lipids is
monitored after collision-induced dissociation. For example, a lipid profile of a class of lipid molecular species can
be obtained by monitoring an ion from the head group of a
class of lipids. Alternatively, a subgroup of lipids containing a common esterified fatty acyl chain can be profiled by
monitoring this fatty acyl carboxylate anions in negativeion mode. Similarly, detection of the loss of a common
neutral fragment after collision-induced dissociation from
their pseudomolecular ions can be used to spectrometrically isolate a class of lipids if the neutrally lost
fragment carries head group information or a subgroup of
lipids if the neutrally lost fragment is from an aliphatic
chain. This technique was first introduced by Cole & Enke
(1991) to detect specific glycerophospholipid classes with
FAB mass spectrometry. For example, choline-containing
phospholipids including both PC and SM were identified
by the PI scanning of m/z 184 corresponding to the protonated phosphocholine moiety present in positive-ion
mode. By ESI tandem mass spectrometry, Smith, Snyder,
& Harden (1995) were able to profile bacterial phospholipids in a great detail.
Tandem mass spectrometric analysis not only can be
used to efficiently characterize lipid classes and individual
molecular species, but also to eliminate baseline noise
(which is frequently seen in mass spectrometric analysis
of low abundance species) and thus greatly simplify the
mass spectrum, allowing one to focus on a single class
or subgroup of lipid. These two advantages make tandem
mass spectrometric technique a candidate for quantitative
analyses of cellular lipidomes if appropriate justifications
have been made (see below). However, caution must be
exercised since tandem mass spectrometric analysis is
also substantially dependent on collisional activation
energy and the structure of individual molecular species.
Therefore, it is difficult to accurately quantitate lipids from
tandem mass spectrometric analysis. To overcome this
issue, Brugger et al. (1997) developed a method by ESI
tandem mass spectrometry using multiple internal standards that cover the range of structural variants in the
biological mixture to quantitatively analyze lipid molecular species. This methodology has recently been adopted
by multiple investigators to quantitate cellular lipidome
of biological samples (e.g., Duffin et al., 2000; Blom et al.,
2001; Welti et al., 2002; Ekroos et al., 2002a; Welti &
Wang, 2004).
The advantages of using tandem mass spectrometric
approach for the analysis of cellular lipidome are apparent

C. Quantitation of Cellular Lipidome by FTMS

Because of its high resolving power, Fourier transform


mass spectrometry (FTMS) is an excellent instrument for
lipid analyses similar to that being used in other research
areas (McLafferty et al., 1995). For example, over 100 lipid
molecular species from mass spectra that were acquired
from both positive- and negative-ion modes have been
readily identified from lipid extracts of biological samples
(Fridriksson et al., 1999; Ivanova et al., 2001). For quantitative analysis of lipids, these investigators first determined a set of relative sensitivities of detection of different
phospholipid classes by using an equimolar mixture of
PC/PE/SM/lysoPC/DAG standards in the positive-ion
mode and a mixture of equimolar PE/PtdIns/PtdSer/
PtdH/PtdGro standards in the negative-ion mode. PE
intensities observed in both ion modes were used to
normalize the spectra with respect to each other. Then, they
determined the relative lipid composition of lipid extracts
by using this set of relative sensitivities. As discussed
above, ionization sensitivities of lipids in a mixture are
mainly dependent on the nature of head groups, but sample
preparation (e.g., pH condition) can dramatically affect
the ionization efficiency of a class of lipids and alter the
relative sensitivities of different lipid classes in a lipid
mixture. Therefore, if the selected standards are added
during the sample preparation and used as internal standards, not only absolute amounts of lipid mass (relative to
protein content or other parameter(s)) can be obtained but
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also the effects of environment factors on quantitative


analysis of lipid molecular species can be minimized.
Furthermore, the factors affecting the quantitative analysis
of lipids such as the lipid concentration in the infused
solution, ion suppression effects, and correction for 13C
isotope factors also need to be considered (see above).

detect and quantitate 1-palmitoyl-2-stearoyl-3-phosphatidylserine in human blood.


Regardless of the synergistic characteristics of HPLC
and mass spectrometry and the aforementioned developments in HPLC-ESI/MS, quantitative analysis of global
cellular lipidome by HPLC-ESI/MS still faces many
challenges. Specifically, when a normal-phase HPLC
column is employed to separate individual lipid classes,
there exists a substantial tailing effect of each individual
peaks because of the presence of hundreds of individual
molecular species, resulting in an uneven distribution of
lipid molecular species across a peak (Taguchi et al., 2000;
Delong et al., 2001). This tailing phenomenon forces
researchers to average all acquired spectra across a peak to
obtain a profile for a specific class. Thus, compared to
intrasource separation, quantitation by HPLC-ESI/MS is
complicated and offers little advantage.
When HPLC is used to separate lipid classes as an
offline device, this tailing phenomenon may cause severe
and differential loss of some molecular species. For
example, if a UV detector is used to monitor the peak
separation of lipids for offline collection, saturated molecular species will be readily underestimated because these
species have a lower UV absorption than unsaturated
species and are enriched in the front of a peak (Gross &
Sobel, 1980). In addition to the tailing effect of HPLC
system, differential loss of lipid molecular species on silica
columns was also observed (Delong et al., 2001). Finally, it
has long been recognized that substantial silica-catalyzed
isomerization and hydrolysis occurs during column
chromatography likely resulting from the well recognized
ability of silica to act as an acid catalyst. Indeed, synthetic
chemists have used silica as an acid catalyst for nearly a
century. HPLC has its advantages for examining very low
abundance analytes that are present in less than one part per
ten thousand. However, because of the multiplicity of
chemistries catalyzed by silica based chemistries, differential elution times of individual molecular species and
hydraulic difficulties as well as surface phenomena, HPLC
must be used with caution in quantitative lipidomics.
When a reversed-phase column is used to resolve
molecular species from a lipid extract of a biological
sample, overlapping of molecular species from different
lipid classes will be very severe, thus potentially resulting
in ion suppression. In addition, the molecular species
of a specific lipid class could elute in an entire run, thus
quantitation is almost impossible if one does not assume
the presence of a linear relationship between the ion
count and the amount of lipids. In practice, it has been
demonstrated by multiple independent studies that this
linear relationship is only held for a low concentration
range of lipid solution (Han & Gross, 1994; Kim, Wang, &
Ma, 1994; Lehmann et al., 1997; Delong et al., 2001;
Koivusalo et al., 2001). Collectively, HPLC-ESI/MS or

D. Quantitation of Cellular Lipidome


by a HPLC-ESI/MS Approach

The combination of HPLC resolving power of lipid classes


(e.g., Gross & Sobel, 1980) and individual molecular
species (e.g., Gross, 1984, 1985; Creer & Gross, 1985) in
conjunction with ESI/MS as the most sensitive and discriminative detector allows these combined approaches to see
even the least abundant lipid molecular species. Gross
(1984, 1985) recognized the combined power of HPLC and
mass spectrometry to perform the initial lipidome analyses
of biological tissues. However, the sensitivity of FAB/MS
was not sufficient to exploit the power of mass spectrometry fully. With the pioneering work of Fenn et al. (1989),
the utility of mass spectrometry for lipid analysis could be
realized. Kim, Wang, & Ma (1994) recognized the power
of the combined HPLC and ESI/MS approach at the very
early stages of its development of ESI/MS for lipid
analyses. The most dramatic example of the power of this
approach has been demonstrated later by Taguchi and
colleagues (Taguchi et al., 2000; Ishida et al., 2001).
By combining HPLC retention time with ESI/MS, they
were able to identify over 500 phospholipid molecular
species from a single preparation of lipid extract from
mouse B cell myeloma NS-1 cells (Taguchi et al., 2000).
Another notable development in LC/MS is the dual parallel
ESI and APCI MS and MS/MS for the analysis of lipids
including phospholipids and TAGs (Byrdwell, 1998, 2003;
Byrdwell & Neff, 2002).
In quantitative analysis of cellular lipidome by using
HPLC-ESI/MS, it is worthwhile to note that Kim, Wang, &
Ma (1994) developed an approach by analyzing either
protonated choline-containing phospholipids or sourceinduced fragments of PE, PtdSer, PtdIns, and PtdH after
reversed-phase HPLC separation. This methodology has
recently been applied to broader biochemical studies (e.g.,
Hamilton et al., 2000; Kevala & Kim, 2001; Kim, Akbar, &
Kim, 2001; Igbavboa et al., 2002; Murthy et al., 2002).
Other investigators have applied the HPLC-ESI/MS or
ESI/MS/MS to quantitate a specific class or group of lipid
molecular species. For example, Sullards and colleagues
(Sullards, 2000; Sullards et al., 2003) used multiple
reaction monitoring (MRM) in conjunction with HPLC
to quantitate sphingolipids including SM, glucosylceramide, ceramide, sphingosine, and sphingosine 1-phosphate. Hvattum et al. (1998) used normal-phase liquid
chromatography coupled with ESI/MS to specifically
30

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HPLC-ESI/MS/MS approach may have tremendous advantages for quantitative analysis of some specific class or
group of lipid molecular species. For quantitative analysis
of global cellular lipidome, this approach needs further
development.

of ESI mass spectrometry to lipidome analyses, this section will give a few examples to illustrate the utility and
its power of this technology in the initial global analyses of cellular lipidome directly from lipid extracts of
biological samples under normal and/or pathological
conditions.

VII. EXAMPLES OF LIPIDOME ANALYSIS UNDER


NORMAL AND PATHOLOGICAL CONDITIONS

A. Alterations in Individual Phospholipid


Molecular Species of Human Platelets
During Thrombin Stimulation

Multiple diseases prevalent in the 21st century resulting


largely from disorders of lipid homeostasis including
diabetes, atherosclerosis, and obesity now afflict industrialized nations (e.g., Unger, 2002). Following recent
excitement about genomics and proteomics, lipidomics
now also is receiving national attention. The recent applications of ESI mass spectrometry in lipid analyses have
underscored the utility of these approaches. Particularly,
the majority of recent ESI/MS applications have shifted
toward the quantitative analysis of lipidomes from biological samples. The applications of ESI/MS in lipid studies
has varied from targeted lipidome analysis (e.g., Ravandi
et al., 1996; Byrdwell & Borchman, 1997; Chen, 1997;
Cheng, Gross, & Pittenauer, 1998; Gunnarsson et al., 1998;
Hvattum et al., 1998; Grullich et al., 2000; Hansen et al.,
2000; Sullards, 2000; Baker et al., 2001; Desai et al., 2002;
Kalderon et al., 2002; Liebisch et al., 2002; Berry et al.,
2003) to global cellular lipidome quantitation (e.g.,
Han et al., 1996, 2000; Blom et al., 2001; Ivanova et al.,
2001; Han & Gross, 2003); from lipid oxidation studies
(e.g., Watson et al., 1999; Harrison et al., 2000; Podrez
et al., 2002; Hoff et al., 2003; Spickett, 2003; Thukkani
et al., 2003) to cell remodeling research (e.g., Brugger
et al., 2000; Kevala & Kim, 2001; Asai et al., 2003); from
the analyses of subcellular membrane fractions (e.g.,
Hoischen et al., 1997; Schneiter et al., 1999; Williams,
Hsu, & Ford, 2000; Pike et al., 2002; Schuck et al., 2003;
Yang, Han, & Gross, 2003) to the examination of transgenic mouse models (e.g., Postle et al., 1999; Hsu et al.,
2000a; Cutler et al., 2002; Finck et al., 2002, 2003; Han
et al., 2002; Dombrowsky et al., 2003; Esch et al., 2003;
Mancuso et al., 2003; Newberry et al., 2003); from studies
on the normal conditions (e.g., Sweetman et al., 1996;
Borrett et al., 1998; Kostiainen et al., 1998; Ramanadham
et al., 1998; Goodacre, Heald, & Kell, 1999; Fang,
Barcelona, & Semrau, 2000; Qiu et al., 2000; Rodgers
et al., 2000; Hsu et al., 2000b) to the investigations on the
pathological or pathophysiological states (e.g., Han et al.,
1996, 2000, 2002; Duffin et al., 2001; Drobnik et al., 2003;
Kakela, Somerharju, & Tyynela, 2003; Listenberger et al.,
2003); and from the research on cancer (e.g., Brachwitz
et al., 1997; Xie et al., 2002) to the studies of Alzheimers
disease (e.g., Han et al., 2002, 2003a,b; Cheng et al., 2003).
Rather than attempting to cover all of the applications

The role of biologically active eicosanoid metabolites as


second messengers in biological processes is well known.
The rate-determining step in the production of these
metabolites in human platelets is the availability of nonesterified AA that is released from endogenous phospholipid storage pools after thrombin stimulation leading a
rapid release of AA in human platelets and enabling the
production of eicosanoid metabolites based upon a cells
precise genetically determined complement of eicosanoid
oxidative enzymes. The first attempt to perform total
lipidome analysis using ESI/MS directly from crude lipid
extracts of biological samples was to examine the lipid
alterations during human platelet activation induced by
thrombin (Han et al., 1996). The purpose of the study was
to investigate the amount of AA mass mobilized and the
source of the released AA during human platelet activation
induced by thrombin stimulation. Thus, we exploited the
analytical power and sensitivity of ESI/MS to profile the
lipid alterations of human platelets during stimulation.
We found that phospholipid masses of all major classes
were dramatically reduced (Fig. 18 and Table 3). A total of
60.1 nmol of AA-containing phospholipids/109 platelets
was hydrolyzed after 90 s after thrombin stimulation.
These hydrolyzed AA-containing phospholipids included
31.8 nmol/109 platelets from the PE pool but only
10.9 nmol/109 platelets from the PC depot. The PtdIns
and PtdSer pools each contributed 8.7 nmol AA-containing
species/109 platelets after thrombin stimulation. We found
most mass lost (63 mol%) from the PE pool was from the
hydrolysis of PlsEtn. This study indicated that (1) PlsEtn
are the largest endogenous pool of AA in human platelets,
(2) loss of PlsEtn molecular species represents the largest
source of AA mass that was mobilized in human platelets after thrombin stimulation, (3) hydrolysis of PtdSer
molecular species accounts for previously-unrecognized
amounts of AA mass that was released during thrombin
stimulation of human platelets, and (4) the lower boundary
for the rate of thrombin-induced AA mobilization in
human platelets can be set >40 nmol/109 platelets/s.
To demonstrate the accuracy in quantitative analysis
by ESI/MS using intrasource separation technique for the
first time, we also assessed the alteration in phospholipids of human platelets during thrombin stimulation by
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A
FIGURE 18. Negative-ion ESI-MS of ethanolamine glycerophospholipids in resting and thrombin-

stimulated human platelets. A: A negative-ion ESI mass spectrum of membrane extract (from resting human
platelets) in the presence of NaOH in 1:2 chloroform/methanol. B: A corresponding negative-ion ESI mass
spectrum of membrane extract from thrombin-stimulated human platelets was acquired under the identical
conditions. The membrane extracts of human platelets were prepared by a modified Bligh & Dyer (1959)
method. The extract, diluted with 1:2 chloroform/methanol in the presence of excess NaOH (i.e., NaOH/
lipid molar ratio >1), was infused directly into the ESI chamber with a syringe pump at a flow rate of 1.5 mL/
min for mass analyses. All indicated individual ethanolamine glycerophospholipid molecular species were
identified using ESI tandem mass spectrometry. (Reprinted from Han et al. (1996) with permission from
American Chemical Society, Copyright 1996.)

conventional analytical methodology (Han et al., 1996).


Specifically, phospholipid classes in lipid extracts of
human platelets were separated by TLC, hydrolyzed by
phospholipase C, and benzoylated. Individual molecular
species of each class were separated by reverse-phase
HPLC (Fig. 19). Each resolved peak was identified by
capillary gas chromatography and quantitated by comparisons of the integrated areas from absorbance profiles to
those of internal standards at 230 nm. Similar to the ESI/
MS analysis, HPLC methodology also demonstrated that
PE pool was predominantly comprised of PlsEtn molecular
species (55%) that contained AA at the sn-2 position
(Fig. 19A and Table 4). After thrombin stimulation, human
platelets lost nearly half of their PE mass (Table 4) and
32

nearly a fifth of their PC mass [Table 2 in original ref.


(Han et al., 1996)]. These results are qualitatively similar
to the results obtained from ESI/MS analysis. The quantitative difference between the two techniques is largely
because of the fact that reverse-phase HPLC can not be
used to accurately quantitate minor constituents and that
peaks that were thought to represent single molecular
species (by their symmetry) actually contained multiple
different molecular species. Collectively, this study not
only resolved the long-time controversy issues regarding
human platelet activation induced by thrombin stimulation
but also clearly demonstrated the accuracy and power
of ESI/MS with intrasource separation technique as an
analytical method in biological studies.

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TABLE 3. Alterations in phospholipid mass in different phospholipid pools during thrombin stimulation of human platelets
quantified by electrospray ionizations mass spectrometrya

B. Lipid Alterations in Diabetic Myocardium

It has now been demonstrated by many investigators that


alterations in cellular lipid homeostasis are associated with
the onset and severity of such diverse diseases as obesity,
atherosclerosis, stroke, and diabetes now epidemic in industrialized nations (see Unger & Orci, 2001; Unger, 2002
for recent reviews). We have exploited ESI intrasource
separation techniques to perform the detailed analysis of
lipidome alterations in rat myocardium rendered diabetic
by streptozotocin treatment (a well-accepted model of
Type-I diabetes) (Han et al., 2000). Negative-ion ESI mass
spectra in the presence of LiOH revealed that myocardial
PlsEtn species in streptozotocin-treated rats were a total of
44% higher than the levels in untreated controls (Fig. 20B
compared to 20A). In contrast, the identical mass spectra
demonstrated that a 22% decrease in 18:0-20:4 PtdEtn
specifically occurred in the PtdEtn subclass (Fig. 20B
compared to 20A) in the absence of a reduction of nonesterified AA or total myocardial AA mass content. Such
ratiometric comparisons are especially valuable when
examining multidimensional spectra for comparisons between control and disease states. Analysis of the identical
myocardial lipid extracts in negative-ion mode in the

a
The cumulative amounts of phospholipids in each of the major
phospholipid pools (by class and by subclass) are expressed in nmol/109
platelets. Change represents the percentage decrement of the phospholipid mass after thrombin stimulation.

FIGURE 19. Reverse-phase HPLC of monobenzoate derivatives of ethanolamine glycerophospholipids in

resting and thrombin-stimulated human platelets. Human platelet ethanolamine glycerophospholipids from
control (A) and thrombin-stimulated (B) human platelets were purified, hydrolyzed by Bacillus cereus
phospholipase C and derivatized. Molecular species were separated by reverse-phase HPLC utilizing an
octadecyl silica column employing acetonitrile/isopropanol (85/15, v/v) as the mobile phase. Major peaks
quantified include the monobenzoate derivatives of 18:3-18:3 PtdEtn (1) (internal standard); 18:120:4 PtdEtn (2); 16:0-20:4 PtdEtn (3); 18:1-20:4 PlsEtn (4); 16:0-20:4 PlsEtn (5); 18:0-20:4 PtdEtn (6); and
18:0-20:4 PlsEtn (7). (Reprinted from Han et al. (1996) with permission from American Chemical Society,
Copyright 1996.)

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TABLE 4. Alterations in ethanolamine glycerophospholipid mass during thrombin stimulation of human plateletsa

a
The data are reprinted with permission from Han et al. (1996), Copyright 1996 American Chemical society. The results
were determined by either ESI/MS or HPLC, expressed in nmol/109 platelets and represent X  S.E. of six separate
experiments. D (diacyl) and P (plasmenyl) indicate PtdEtn and PtEtn molecular species, respectively.
b
Several minor species corresponding to the alkyl ether species (m/z 703, 721, 731, 751, and 753) were also found which
persisted even after the samples were treated with acidic vapor. Since these species were of insufficient mass (<0.5% each) to
perform tandem mass spectrometic analyses to confirm their identity, they have not been included in the table.
c
The mass of all ions was rounded to the nearest integer.
d
Arachidonic acid-containing phosphollpids.

absence of LiOH demonstrated a 46% increase in myocardial PtdIns mass in diabetic rats in comparison to
normal controls (Fig. 20D compared to 20C). Although no
alterations in diabetic myocardial PC content were found,
positive-ion ESI mass spectra of myocardial lipid extracts
34

in the presence of LiOH revealed that a massive remodeling of TAG molecular species occurred including an
over 5-fold increase in tripalmitin mass and a 60% decrease
in TAG molecular species containing polyunsaturated acyl
acids in diabetic rat myocardium (Han et al., 2000). ESI

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A
FIGURE 20. Representative ESI mass spectra of myocardial lipid extracts from control and diabetic rats.

Negative-ion ESI mass spectra of myocardial lipid extracts from control (spectra A and C) and diabetic
(spectra B and D) rats were acquired in the presence of LiOH (spectra A and B which show PE molecular
species) and absence of LiOH (spectra C and D which show anionic phospholipids). Individual molecular
species were identified using tandem mass spectrometry. The internal standards were 15:0-15:0 PtdEtn (m/z
663) or 14:0-16:0 PtdGro (m/z 694). The masses of all ions were rounded to the nearest integer. The clusters
of m/z 723, 747, and 775 in spectra A and B are corresponding to PlsEtn molecular species. (Reprinted from
Han et al. (2000) with permission from Biochemical Society, Copyright 2000.)

mass spectroscopic analysis further demonstrated the complete recovery of each of the diabetes-induced alterations
in phospholipid classes, subclasses, and individual molecular species after peripheral insulin treatment. In sharp
contrast, the substantial alterations in TAG molecular
species were not prevented by insulin treatment after induction of diabetic state. These results indicated that
dramatic lipid alterations in myocardial lipid metabolism
are associated with the diabetic state and that these alterations cannot be recovered by routine insulin administration
alone. Accordingly, this lipidomic study has already led to
new insights into the mechanisms underlying the diabetic
state and the importance (and potential toxicity) of saturated vs. non-saturated fatty acids and their respective
metabolic fates.

C. Specific Lipid Changes in the Very


Early Stages of Alzheimers Disease

Recently, the power of the lipidomics using ESI intrasource separation techniques has been applied to the
studies on the pathological mechanism(s) underlying
Alzheimers disease (AD) (Han, Holtzman, & McKeel,
2001; Han et al., 2002). It was unambiguously demonstrated for the first time that very distinct profiles of PE
molecular species in lipid extracts of human brain gray
matter and white matter were present (Fig. 21A compared
to 21B) (Han, Holtzman, & McKeel, 2001). These distinct
lipid profiles between gray matter and white matter indicate the distinct structure and cellular function relationship of these basic tissue compartments. Remarkably, the
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A
FIGURE 21. Representative negative-ion ESI mass spectra of ethanolamine glycerophospholipids from

temporal gray and white matters of normal human and AD brains. Brain samples were obtained from the
brain bank of the Washington University ADRC Neuropathology/Tissue Resource Core and lipids were
extracted by a modified method of Bligh & Dyer (1959). Negative-ion ESI mass spectra of lipid extracts of
temporal gray matter (Panel A) and white matter (panel B) of normal human brain, or temporal white matter
of AD brain (panel C) were acquired in the presence of LiOH. Individual PE molecular species were
identified using tandem mass spectrometry. The internal standard (I.S.) is 15:0-15:0 PtdEtn (m/z 662.6).
(Reprinted from Han et al. (2001) with permission from International Society for Neurochemistry,
Copyright 2001.)

distinct mass spectral profiles provide an efficient criterion to demonstrate the purity of gray and white matter.
These well-defined differences in lipid profiles were
impossible to obtain with other lipid analytical methodologies and clearly demonstrate the power of lipidomics by
ESI/MS.
36

In those studies, it was found that three specific


alterations in lipids were present at the earliest clinically
recognizable stage of AD (i.e., mild cognitive impairment
because of AD). First, a dramatic reduction of PlsEtn mass
(up to 40 mol% of total plasmalogens) in both gray matter
and white matter was present in AD subjects in compari-

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son to those age-matched cognitively normal controls


(e.g., Fig. 21C compared to 21B; Most of the major
PlsEtn molecular species are indicated in the figures)
(Han, Holtzman, & McKeel, 2001). These results imply
a potential mechanism of oxidation in AD pathogenesis
(Markesbery, 1997). Second, sulfatides, a specialized component of the myelin sheath, are depleted up to 93 and

58 mol% of total sulfatide mass content in gray matter and


white matter, respectively, in all examined brain regions
from AD subjects with very mild dementia (e.g., Fig. 22,
Han et al., 2002). Further studies demonstrated that
apolipoprotein E modulates the sulfatide content in the
central nervous system (Han & Gross, 2003), whereas E4
allele of apolipoprotein E is the only known risk factor for

A
FIGURE 22. Representative negative-ion ESI mass spectra of lipid extracts from temporal gray matter

from a subject who was cognitively normal and a subject with very mild Alzheimers type dementia. Lipids
were extracted by a modified method of Bligh & Dyer (1959) and negative-ion ESI mass spectra of lipid
extracts of temporal gray matter of cognitively normal human brain (panel A), or of AD brain (panel B)
were acquired in the absence of LiOH. All major individual molecular species as indicated were identified
using tandem mass spectrometry. (Reprinted from Han et al. (2002) with permission from International
Society for Neurochemistry, Copyright 2002.)

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late-onset AD (Strittmatter & Roses, 1996; Weisgraber &


Mahley, 1996). The third specific alteration in lipidome
of AD subjects is the dramatic elevation (>3-fold) of
ceramide content in white matter of all examined regions
(Han et al., 2002). Accumulation of ceramide content can
lead to diverse biological consequences, such as upregulation of cytokines, generation of reactive oxygen
species, interruption of the mitochondrial respiratory
chain, apoptosis, etc. (Hannun, 1996; Kolesnick & Kronke,
1998; Paola, Cocco, & Lorusso, 2000). Therefore, accumulation of ceramide content itself could contribute to
the neurodegeneration that occurs in AD. No alterations in
other examined lipids including PtdEtn, PtdCho, SM,
GalC, PtdIns, PtdGro, and PtdSer are present at the earliest
clinically recognizable stage of AD (e.g., Fig. 22) and only
modest reduction (15 mol%) of these lipids occurs at the
very severe stage of AD (Han, Holtzman, & McKeel, 2001;
Han et al., 2002). Accordingly, these specific alterations in
the lipidome may play an important role in the pathogenesis of AD and be linked with early events in the
pathological process of AD including neurodegeneration,
synapse loss, synaptic dysfunction and progressive memory loss in AD.

D. The Role of Phospholipase Da


a in Plant Lipid
Alterations Under Freezing Stresses

Plant stress caused by freezing at a sublethal temperature


has been an area of intense research, but the molecular
and cellular mechanisms of freezing-induced injury and
tolerance still remain elusive. Membrane lipid changes that
occur during freezing stress may play a role in injury and/
or tolerance. Phospholipase D (PLD), which hydrolyzes
phospholipids to PtdH, is particularly abundant in plants.
Therefore, Welti et al. (2002, 2003) applied the ESI
tandem mass spectrometric approach as first proposed
by Brugger et al. (1997) to explore the lipid alterations
in Arabidopsis undergoing cold and freezing stresses.
Welti et al. (2003) developed multiple tandem mass
scan conditions that were specific for chloroplast lipids
including monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG). Both MGDG and DGDG in the plant
lipid extracts were profiled and quantitated by PI scanning
of m/z 243 (corresponding to a sodiated galactose derivative) from the sodium-adducted molecular ions in the
presence of multiple internal standards (Fig. 23A,B). Similarly, the SQDG molecular species in plant lipid extracts
were analyzed by PI scanning of m/z 225 (corresponding to
a galatose derivative) from the deprotonated molecular
ions in negative-ion mode (Fig. 23C).
By employing the ESI tandem mass spectrometric
analyses in either PI or NL mode, Welti et al. (2002)
profiled and quantitated the molecular species of plant
38

FIGURE 23. ESI PI scans of individual molecular species of mono-

galactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol


(DGDG), and sulfoquinovosyldiacylglycerol (SQDG) directly from
the crude lipid extract of Arabidopsis. Both MGDG (panel A) and
DGDG (panel B) molecular species were profiled by PI scanning of
m/z 243 from their sodium adducts at a collision energy of 45 and 66 eV,
respectively, in positive-ion mode. SQDG (panel C) molecular
species was profiled by PI scanning of m/z 225 in negative-ion mode
from their deprotonated molecular ions. (Reprinted from Welti, Wang,
& Williams, 2003 with permission from Elsevier Science (USA),
Copyright 2003.)

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A
FIGURE 24. Freezing-induced changes in lipid molecular species in Arabidopsis as determined by ESI

tandem mass spectrometry. The black bars represent cold-acclimated plants and the hatched bars represent
plants subjected to 88C. The values are the means  S.D. (n 4 or 5). A L indicates that the value is
lower than that of the cold-acclimated plants of the same genotype (P < 0.05). A H indicates that the value
is higher than that of the cold-acclimated plants of the same genotype (P < 0.05). Panel A: Phospholipids
and galactolipids of wild-type plants. Panel B: Phospholipids and galactolipids of PLDa-deficient plants.
(Reprinted from Welti et al., 2002 with permission from The American Society for Biochemistry and
Molecular Biology, Inc., Copyright 2002.)

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A
FIGURE 25. Representative positive-ion ESI/FTMS mass spectra of lipid extracts from intact,

permeabilized, and scPLD-treated RBL-2H3 cells. Labels on the peaks indicate phospholipid species
head groups (xx:y, where xx total number of carbon atoms in the fatty acid chains, and y number of
double bonds). (Reprinted from Ivanova et al., (2001) with permission from the National Academy of
Sciences, Copyright 2001).

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A
FIGURE 26. Representative negative-ion ESI/FTMS mass spectra of lipid extracts from intact,

permeabilized, and scPLD-treated RBL-2H3 cells. Labels on the peaks indicate phospholipid species
head groups (xx:y, where xx total number of carbon atoms in the fatty acid chains, and y number of
double bonds). (Reprinted from Ivanova et al. (2001) with permission from the National Academy of
Sciences, Copyright 2001.)

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lipids including PC, PE, PtdGro, PtdIns, PtdSer, PtdH,


lysoPC, lysoPE, MGDG, and DGDG (Fig. 24A). Dramatic
alterations in these plant lipids induced by freezing at
88C were also demonstrated (Fig. 24A). They found that
massive declines in the levels of PC, PE, and PtdGro

occurred in conjunction with increases in the masses of


PtdH, lysoPC, and lysoPE, whereas other lipid classes
(e.g., PtdIns, MGDG, and DGDG) were only modestly
changed. They believe that these results suggest a specific rise in phospholipase activities during freezing.

A
FIGURE 27. Two-dimensional map of LC-ESI/MS of phospholipid mixtures from NS-1 cells. A capillary

Si60 LC column (150  0.3 mm i.d., 5 mm) was used for chromatographic separation of phospholipids.
Panel A: Positive pseudomolecular ion 2-D map of phospholipids obtained at a cone voltage of 30 V; (panel
B) negative pseudomolecular ion 2-D map of phospholipids obtained at a cone voltage of 30 V. (Reprinted
from Taguchi et al. (2000) with permission from John Wiley & Sons, Ltd., Copyright 2000.)

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Specifically, the lower level of lysoPC and lysoPE in


comparison to PtdH indicates that PLD is more active than
other phospholipases in frozen plant tissue (Welti et al.,
2002).
Therefore, to determine the role of PLD in freezinginduced lipid changes in plants, Welti and colleagues
comparatively analyzed lipid alterations in PLDa-deficient
Arabidopsis plants by tandem mass spectrometry
(Fig. 24B) because PLDa is the most common plant
PLD. They demonstrated that, in wild-type and PLDadeficient plants, there was a substantial difference in the
PC decrease (45% vs. 22%) upon freezing at 88C, where
PE and PtdGro decreases were similar in wild-type and
mutant plant. During freezing, PtdH levels in PLDadeficient plants were increased only 50% as much as the
levels in wild-type plants. These results suggest that PC is

the major in vivo substrate of PLDa and this enzyme is


responsible for approximately half of PtdH production.

A
E. Alterations in Phospholipids in Mast
Cells During Degranulation

Mast cells contribute to immune and allergic response by


release of secretory granule contents (i.e., degranulation)
after a complex biochemical cascade initiated by antigens.
One step of the biochemical cascade is the production of
lipid second messengers such as PtdH by PLD, DAG by
phospholipase C, and free fatty acid and lysoPC by PLA2.
However, Cohen & Brown (2001) found that degranulation in RBL-2H3 cells, a mucosal mast cell line, can also
be induced in vitro by addition of multiple exogenous
phospholipases (all of which were reported to hydrolyze

FIGURE 28. Positive-ion mass spectra of choline glycerophospholipids at different elution times in

capillary LC. Panel A: Expanded 2-D map of PC in Figure 27. Panel B: Mass spectra of PC at different
elution times. (Reprinted from Taguchi et al. (2000) with permission from John Wiley & Sons, Ltd.,
Copyright 2000.).

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A
FIGURE 28. (Continued )

PC preferentially) without activating intracellular-signaling pathways. To determine the changes in lipid classes
and molecular species in these processes, Brown and
Colleagues (Ivanova et al., 2001) performed lipid analyses
of cellular lipid extracts by using FTMS in both positiveand negative-ion modes.
The positive- and negative-ion ESI/FTMS mass
spectra of lipid extracts from intact, permeabilized, and
PLD-treated RBL-2H3 cells are shown in Figures 24
and 25, respectively. One of the most prominent differences found from the positive-ion FTMS analyses was the
altered molecular species composition of DAGs (Fig. 25).
Only two or three species with short saturated or short
polyunsaturated alkyl chains were present in intact or
permeabilized cells, whereas DAG species with longer
44

(18 or more C atoms) polyunsaturated alkyl chains were


predominant in PLD-treated cells. In addition, after treatment with PLD from Streptomyces chromofuscus (sc), the
PC molecular species containing long (20 C atoms) and
polyunsaturated alkyl chains disappeared (Fig. 25). In
contrast to the disappearance of PC species, SM species
seem to be synthesized because of the increase in the
availability of phosphocholine from the scPLD-induced
PC hydrolysis. Mass spectrometric analyses in negativeion mode (Fig. 26) did not identify any dramatic changes in
mass of PtdIns, PtdGro, and PtdSer although the mass of
both saturated and unsaturated PtdH species were
substantially increased. These studies resolved over 130
different glycerophospholipid molecular species, quantitated the relative lipid alterations induced by exogenous

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phospholipases, and found PC species were the preferred


substrates for the enzymes.

soon be revealed in no small part by the power of mass


spectrometry.

F. Analysis of Phospholipid Molecular Species


Directly From Cellular Lipid Extracts
by HPLC-ESI/MS

IX. ABBREVIATIONS

Recently, Taguchi et al. (2000) developed a pseudo 2D


method to directly map phospholipid molecular species
of cellular lipid extracts, which was constructed by
employing the elution time in the first dimension and
using molecular mass as the second dimension. By using a
quadrupole mass spectrometer, the authors were able to
map phospholipid species with four different functions
including positive molecular ions (Fig. 27A), negative
molecular ions (Fig. 27B), positive fragment ions, and
negative fragment ions. The map of positive molecular ions
demonstrated molecular species from the classes of PE,
PC, SM, and lysoPC (Fig. 27A). The map of negative
molecular ions showed the molecular species of PtdIns,
PE, PC, SM, and lysoPC (Fig. 27B). The head groups
and aliphatic chains of these molecular species were recognized in the maps of positive and negative fragment
ions that were induced in the ion source. From this
comprehensive analysis, more than 500 molecular species
of phospholipids were identified within a few hours immediately after extraction from the culture cells. In addition,
Taguchi and colleagues demonstrated that the prominent
PC molecular species were in the plasmanylcholine subclass over plasmenylcholine subclass in mouse B cell
myeloma NS-1 cells (Fig. 28) as confirmed with mild acid
treatment, whereas predominant molecular species of
plasmalogen subclass were present in PE. As demonstrated
(Fig. 28), the distribution of molecular species crossing
each peak is not homogenous, thus, no quantitative results
for individual molecular species could be made from the
study.

AA
AD
DAG
ESI
FAB
FTMS
GalC
m:n

VIII. CONCLUSION

ACKNOWLEDGMENTS

The development of ESI/MS by Fenn and colleagues


in conjunction with previously developed lipid HPLC
methods, mass spectrometric approaches, and critical lipid
chemical methods has dramatically advanced the field of
lipidomics. Multiple quantitative approaches based on
ESI/MS have been developed and used to identify and
quantitate molecular constituents in cellular lipidomes.
The applications of these techniques are broad, but the
limitations of each approach are also obvious. These techniques will serve as a template for further progress and
development in the field. It is clear that the power of
lipidomics is just beginning to be realized and that the
biochemical mechanisms underlying disease states will

MS
NEFA
NL
Nm:n

PC
PE
PI
PlsEtn
PtdCho
PtdEtn
PtdGro
PtdH
PtdIns
PtdSer
SM
TAG
Tm:n TAG

arachidonic acid
Alzheimers disease
diacylglycerols
electrospray ionization
fast atom bombardment
Fourier transform mass spectrometry
galactocerebroside(s)
acyl chain containing m carbons and n
double bonds
mass spectrometry
non-esterified fatty acid(s)
neutral loss
acyl amide with m carbons and n double
bonds
choline glycerophospholipids
ethanolamine glycerophospholipids
precursor ion
plasmenylethanolamine(s)
phosphatidylcholine(s)
phosphatidylethanolamine(s)
phosphatidylglycerol(s)
phosphatidic acid(s)
phosphatidylinositol(s)
phosphatidylserine(s)
sphingomyelin(s)
triacylglycerol(s)
triacylglycerol with identical acyl chains
each containing m carbons and n double
bonds

Xianlin Han is the 2003 Memory Ride Prize recipient.


The authors are thankful to the Washington University
Mass Spectrometer Facility Center for the use of electrospray ionization mass spectrometer. The authors are
thankful to Dr. Fong-Fu Hsu for his comments.

REFERENCES

Acharya U, Patel S, Koundakjian E, Nagashima K, Han X,


Acharya JK. 2003. Modulating sphingolipid biosynthetic
pathway rescues photoreceptor degeneration. Science 299:
17401743.
45

&

HAN AND GROSS

Author Proof

Alessenko AV, Burlakova EB. 2002. Functional role of phospholipids in the nuclear events. Bioelectrochemistry 58:
1321.
Asai K, Hirabayashi T, Houjou T, Uozumi N, Taguchi R,
Shimizu T. 2003. Human group IVC phospholipase A2
(cPLA2gamma). Roles in the membrane remodeling and
activation induced by oxidative stress. J Biol Chem 278:
88098814.
Ashcroft SJH. 1997. Intracellular second messengers. Adv Exp
Med Biol 426:7380.
Athenstaedt K, Daum G. 1999. Phosphatidic acid, a key intermediate in lipid metabolism. Eu J Biochem 266:116.
Baker DL, Umstot ES, Desiderio DM, Tigyi GJ. 2000. Quantitative analysis of lysophosphatidic acid in human blood
fractions. Ann NY Acad Sci 905:267269.
Baker DL, Desiderio DM, Miller DD, Tolley B, Tigyi GJ. 2001.
Direct quantitative analysis of lysophosphatidic acid
molecular species by stable isotope dilution electrospray
ionization liquid chromatography-mass spectrometry. Anal
Biochem 292:287295.
Beckedorf AI, Schaffer C, Messner P, Peter-Katalinic J. 2002.
Mapping and sequencing of cardiolipins from Geobacillus
stearothermophilus NRS 2004/3a by positive and negative
ion nanoESI-QTOF-MS and MS/MS. J Mass Spectrom
37:10861094.
Berry KAZ, Borgeat P, Gosselin J, Flamand L, Murphy RC. 2003.
Urinary metabolites of leukotriene B4 in the human subject.
J Biol Chem 278:2444924460.
Bligh EG, Dyer WJ. 1959. A rapid method of total lipid
extraction and purification. Can J Biochem Physiol 37:911
917.
Blom TS, Koivusalo M, Kuismanen E, Kostiainen R, Somerharju
P, Ikonen E. 2001. Mass spectrometric analysis reveals an
increase in plasma membrane polyunsaturated phospholipid
species upon cellular cholesterol loading. Biochemistry
40:1463514644.
Bocckino SB, Exton JH. 1996. Phosphatidic acid. Handbook
Lipid Res 8:75123.
Borrett VT, Duncan A, Mathews RJ, Traeger JC. 1998.
Electrospray mass spectrometry of phospholipids extracted
from inactivated bacteria. Adv Mass Spectrom 14:C056070/
1C056070/13.
Brachwitz H, Thomas Y, Bergmann J, Langen P, Berdel WE.
1997. New nucleoside-50 -alkylphosphonophosphates and
related compounds containing 20 -deoxycytidine, thymidine
and adenosine as nucleoside component. Syntheses and their
effects on tumor cell growth in vitro. Chem Phys Lipids
87:3139.
Brugger B, Erben G, Sandhoff R, Wieland FT, Lehmann WD.
1997. Quantitative analysis of biological membrane lipids at
the low picomole level by nano-electrospray ionization
tandem mass spectrometry. Proc Natl Acad Sci USA 94:
23392344.
Brugger B, Sandhoff R, Wegehingel S, Gorgas K, Malsam J,
Helms JB, Lehmann W-D, Nickel W, Wieland FT. 2000.
Evidence for segregation of sphingomyelin and cholesterol

during formation of COPI-coated vesicles. J Cell Biol 151:


507517.
Byrdwell WC. 1998. Dual parallel mass spectrometers for
analysis of sphingolipid, glycerophospholipid and plasmalogen molecular species. Rapid Commun Mass Spectrom
12:256272.
Byrdwell WC. 2003. Dual parallel liquid chromatography/dual
mass spectrometry (LC2/MS2) of bovine brain total lipid
extract. J Liq Chromatogr R T 26:31473181.
Byrdwell WC, Borchman D. 1997. Liquid chromatography/
mass-spectrometric characterization of sphingomyelin and
dihydrosphingomyelin of human lens membranes. Ophthalmic Res 29:191206.
Byrdwell WC, Neff WE. 2002. Dual parallel electrospray
ionization and atmospheric pressure chemical ionization
mass spectrometry (MS), MS/MS and MS/MS/MS for the
analysis of triacylglycerols and triacylglycerol oxidation
products. Rapid Commun Mass Spectrom 16:300319.
Chen S. 1997. Tandem mass spectrometric approach for
determining structure of molecular species of aminophospholipids. Lipids 32:85100.
Cheng C, Gross ML, Pittenauer E. 1998. Complete structural
elucidation of triacylglycerols by tandem sector mass spectrometry. Anal Chem 70:44174426.
Cheng H, Xu J, Mckeel DW Jr, Han X. 2003. Specificity and
potential mechanism of sulfatide deficiency in Alzheimers
disease: An electrospray ionization mass spectrometric
study. Cell Mol Biol 49:809818.
Cohen JS, Brown HA. 2001. Phospholipases stimulate secretion
in RBL mast cells. Biochemistry 40:65896597.
Cole RB. 1997. Electrospray ionization mass spectrometry. New
York: Wiley.
Cole MJ, Enke CG. 1991. Direct determination of phospholipid
structures in microorganisms by fast atom bombardment
triple quadrupole mass spectrometry. Anal Chem 63:1032
1038.
Creer MH, Gross RW. 1985. Reversed-phase high-performance
liquid chromatographic separation of molecular species of
alkyl ether, vinyl ether, and monoacyl lysophospholipids.
J Chromatogr 338:6169.
Cullis PR, Fenske DB, Hope MJ. 1996. Physical properties and
functional roles of lipids in membranes. In: Vance DE,
Vance J, editors. Biochemistry of lipids, lipoproteins
and membranes. Amsterdam, The Netherlands: Elsevier.
pp 133.
Cutler RG, Pedersen WA, Camandola S, Rothstein JD, Mattson
MP. 2002. Evidence that accumulation of ceramides and
cholesterol esters mediates oxidative stress-induced death of
motor neurons in amyotrophic lateral sclerosis. Ann Neurol
52:448457.
de la Mora JF, van Berkel GJ, Enke CG, Cole RB, MartinezSanchez M, Fenn JB. 2000. Electrochemical processes in
electrospray ionization mass spectrometry. J Mass Spectrom
35:939952.
DeLong CJ, Baker PRS, Samuel M, Cui Z, Thomas MJ. 2001.
Molecular species composition of rat liver phospholipids by

46

SHOTGUN LIPIDOMICS BY ESI/MS

&

Author Proof

ESI-MS/MS: The effect of chromatography. J Lipid Res


42:19591968.
Desai K, Sullards MC, Allegood J, Wang E, Schmelz EM, Hartl
M, Humpf H-U, Liotta DC, Peng Q, Merrill AH Jr. 2002.
Fumonisins and fumonisin analogs as inhibitors of ceramide
synthase and inducers of apoptosis. Biochim Biophys Acta
1585:188192.
di Marzo V. 1995. Review: Arachidonic acid and eicosanoids as
targets and effectors in second messenger interactions.
Prostag Leukotr Ess 53:239254.
Dombrowsky H, Clark GT, Rau GA, Bernhard W, Postle AD.
2003. Molecular species compositions of lung and pancreas
phospholipids in the cftr(tm1HGU/tm1HGU) cystic fibrosis
mouse. Pediatr Res 53:447454.
Drobnik W, Liebisch G, Audebert F-X, Frohlich D, Gluck T,
Vogel P, Rothe G, Schmitz G. 2003. Plasma ceramide and
lysophosphatidylcholine inversely correlate with mortality
in sepsis patients. J Lipid Res 44:754761.
Duffin KL, Henion JD, Shieh JJ. 1991. Electrospray and tandem
mass spectrometric characterization of acylglycerol mixtures that are dissolved in nonpolar solvents. Anal Chem
63:17811788.
Duffin KL, Obukowicz MG, Raz A, Shieh JJ. 2000. Electrospray/
tandem mass spectrometry for quantitative analysis of lipid
remodeling in essential fatty acid deficient mice. Anal
Biochem 279:179188.
Duffin KL, Obukowicz MG, Salsgiver WJ, Welsch DJ, Shieh C,
Raz A, Needleman P. 2001. Lipid remodeling in mouse liver
and plasma resulting from delta6 fatty acid desaturase
inhibition. Lipids 36:12031208.
Ekroos K, Shevchenko A. 2002b. Simple two-point calibration
of hybrid quadrupole time-of-flight instruments using a
synthetic lipid standard. Rapid Commun Mass Spectrom 16:
12541255.
Ekroos K, Chernushevich IV, Simons K, Shevchenko A. 2002a.
Quantitative profiling of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight
mass spectrometer. Anal Chem 74:941949.
Ekroos K, Ejsing CS, Bahr U, Karas M, Simons K, Shevchenko
A. 2003. Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation. J Lipid Res 44:21812192.
English D. 1996. Phosphatidic acid: A lipid messenger involved
in intracellular and extracellular signalling. Cell Signal
8:341347.
Esch SW, Williams TD, Biswas S, Chakrabarty A, Levine SM.
2003. Sphingolipid profile in the CNS of the twitcher
(globoid cell leukodystrophy) mouse: A lipidomics
approach. Cell Mol Biol 49:779787.
Fang J, Barcelona MJ, Semrau JD. 2000. Characterization of
methanotrophic bacteria on the basis of intact phospholipid
profiles. FEMS Microbiol Lett 189:6772.
Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. 1989.
Electrospray ionization for mass spectrometry of large
biomolecules. Science 246:6471.

Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. 1990.
Electrospray ionization-principles and practice. Mass Spectrom Rev 9:3770.
Finck BN, Lehman JJ, Leone TC, Welch MJ, Bennett MJ, Kovacs
A, Han X, Gross RW, Kozak R, Lopaschuk GD, Kelly DP.
2002. The cardiac phenotype induced by PPARa overexpression mimics that caused by diabetes mellitus. J Clin
Invest 109:121130.
Finck BN, Han X, Courtois M, Aimond F, Nerbonne JM, Kovacs
A, Gross RW, Kelly DP. 2003. A critical role for PPARamediated lipotoxicity in the pathogenesis of diabetic
cardiomyopathy: Modulation of phenotype by dietary fat
content. Proc Natl Acad Sci USA 100:12261231.
Folch J, Lees M, Stanley GHS. 1957. A simple method for the
isolation an purification of total lipids from animal tissues.
J Biol Chem 226:497509.
Ford DA, Rosenbloom KB, Gross RW. 1992. The primary
determinant of rabbit myocardial ethanolamine phosphotransferase substrate selectivity is the covalent nature of the
sn-1 aliphatic group of diradyl glycerol acceptors. J Biol
Chem 267:1122211228.
Ford DA, Han X, Horner CC, Gross RW. 1996. Accumulation of
unsaturated acylcarnitine molecular species during acute
myocardial ischemia: Metabolic compartmentalization of
products of fatty acyl chain elongation in the acylcarnitine
pool. Biochemistry 35:79037909.
Fridriksson EK, Shipkova PA, Sheets ED, Holowka D, Baird B,
McLafferty FW. 1999. Quantitative analysis of phospholipids in functionally important membrane domains from
RBL-2H3 mast cells using tandem high-resolution mass
spectrometry. Biochemistry 38:80568063.
Ghosh S, Strum JC, Bell RM. 1997. Lipid biochemistry: Functions of glycerolipids and sphingolipids in cellular signaling.
FASEB J 11:4550.
Glaser PE, Gross RW. 1994. Plasmenylethanolamine facilitates
rapid membrane fusion: A stopped-flow kinetic investigation correlating the propensity of a major plasma membrane
constituent to adopt an HII phase with its ability to promote
membrane fusion. Biochemistry 33:58055812.
Glaser PE, Gross RW. 1995. Rapid plasmenylethanolamineselective fusion of membrane bilayers catalyzed by an
isoform of glyceraldehyde-3-phosphate dehydrogenase:
Discrimination between glycolytic and fusogenic roles of
individual isoforms. Biochemistry 34:1219312203.
Goni FM, Alonso A. 1999. Structure and functional properties of
diacylglycerols in membranes. Prog Lipid Res 38:148.
Goodacre R, Heald JK, Kell DB. 1999. Characterization of intact
microorganisms using electrospray ionization mass spectrometry. FEMS Microbiol Lett 176:1724.
Griffiths WJ. 2003. Tandem mass spectrometry in the study of
fatty acids, bile acids, and steroids. Mass Spectrom Rev
22:81152.
Griffiths WJ, Brown A, Reimendal R, Yang Y, Zhang J, Sjoevall J.
1996. A comparison of fast-atom bombardment and electrospray as methods of ionization in the study of sulfated- and

47

&

HAN AND GROSS

Author Proof

sulfonated lipids by tandem mass spectrometry. Rapid


Commun Mass Spectrom 10:11691174.
Gross RW. 1984. High plasmalogen and arachidonic acid content
of canine myocardial sarcolemma: A fast atom bombardment mass spectroscopic and gas chromatography-mass
spectroscopic characterization. Biochemistry 23:158165.
Gross RW. 1985. Identification of plasmalogen as the major
phospholipid constituent of cardiac sarcoplasmic reticulum.
Biochemistry 24:16621668.
Gross RW. 1992. Myocardial phospholipases A2 and their membrane substrates. Trends Cardiovas Med 2:115121.
Gross RW, Sobel BE. 1980. Isocratic high-performance liquid
chromatography separation of phosphoglycerides and lysophosphoglycerides. J Chromatogr 197:7985.
Grullich C, Sullards MC, Fuks Z, Merrill AH Jr, Kolesnick R.
2000. CD95(Fas/APO-1) signals ceramide generation
independent of the effector stage of apoptosis. J Biol Chem
275:86508656.
Gu M, Kerwin JL, Watts JD, Aebersold R. 1997. Ceramide profiling of complex lipid mixtures by electrospray ionization
mass spectrometry. Anal Biochem 244:347356.
Gunnarsson T, Karlsson A, Hansson P, Johnson G, Alling C,
Odham G. 1998. Determination of phosphatidylethanol in
blood from alcoholic males using high-performance liquid
chromatography and evaporative light scattering or electrospray mass spectrometric detection. J Chromatogr B
705:243249.
Hamilton J, Greiner R, Salem N Jr, Kim H-Y. 2000. n-3 Fatty acid
deficiency decreases phosphatidylserine accumulation
selectively in neuronal tissues. Lipids 35:863869.
Han X. 2002. Characterization and direct quantitation of
ceramide molecular species from lipid extracts of biological
samples by electrospray ionization tandem mass spectrometry. Anal Biochem 302:199212.
Han X, Holtzman DM, McKeel DW Jr. 2001. Plasmalogen deficiency in early Alzheimers disease subjects and in animal
models: Molecular characterization using electrospray
ionization mass spectrometry. J Neurochem 77:11681180.
Han X, Gross RW. 1990. Plasmenylcholine and phosphatidylcholine membrane bilayers possess distinct conformational
motifs. Biochemistry 29:49924996.
Han X, Gross RW. 1991. Modulation of cardiac membrane
fluidity by amphiphilic compounds and their role in the
pathophysiology of myocardial infarction. In: Aloia RC,
Curtain CC, Gordon LM, editors. Drug and anesthetic
effects on membrane structure and function. New York:
Wiley-Liss, Inc. pp 225243.
HanQ3 X, Gross RW. 1991. Proton nuclear magnetic resonance
studies on the molecular dynamics of plasmenylcholine/
cholesterol and phosphatidylcholine/cholesterol bilayers.
Biochim Biophys Acta 1063:129136.
Han X, Gross RW. 1994. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane
phospholipids. Proc Natl Acad Sci USA 91:1063510639.
Han X, Gross RW. 1995. Structural determination of picomole
amounts of phospholipids via electrospray ionization

tandem mass spectrometry. J Am Soc Mass Spectrom 6:


12021210.
Han X, Gross RW. 1996. Structural determination of lysophospholipid regioisomers by electrospray ionization tandem
mass spectrometry. J Am Chem Soc 118:451457.
Han X, Gross RW. 2001. Quantitative analysis and molecular
species fingerprinting of triacylglyceride molecular species
directly from lipid extracts of biological samples by
electrospray ionization tandem mass spectrometry. Anal
Biochem 295:88100.
Han X, Gross RW. 2003. Global analysis of cellular lipidomes
directly from crude extracts of biological samples by ESI
mass spectrometry: A bridge to lipidomics. J Lipid Res
44:10711079.
Han X, Gubitosi-Klug RA, Collins B, Gross RW. 1996.
Alternations in individual molecular species of human
platelet phospholipids during thrombin stimulation: Electrospray ionization mass spectrometry-facilitated identification of the boundary conditions for the magnitude and
selectivity of thrombin-induced platelet phospholipid
hydrolysis. Biochemistry 35:58225832.
Han X, Abendschein AR, Kelley JG, Gross RW. 2000. Diabetesinduced changes in specific lipid molecular species in rat
myocardium. Biochem J 352:7989.
Han X, Holtzman DM, McKeel DW Jr, Kelley J, Morris JC. 2002.
Substantial sulfatide deficiency and ceramide elevation in
very early Alzheimers disease: Potential role in disease
pathogenesis. J Neurochem 82:809818.
Han X, Cheng H, Fryer DJ, Fagan AM, Holtzman DM. 2003a.
Novel role for apolipoprotein E in the central nervous
system: Modulation of sulfatide content. J Biol Chem 278:
80438051.
Han X, Fagan AM, Cheng H, Morris JC, Xiong C, Holtzman DM.
2003b. Cerebrospinal fluid sulfatide is decreased in subjects
with incipient dementia. Ann Neurol 54:115119.
Hannun YA. 1996. Function of ceramide in coordinating cellular
responses to stress. Science 274:18551859.
Hannun YA, Luberto C. 2000. Ceramide in the eukaryotic stress
response. Trends Cell Biol 10:7380.
Hansen HH, Hansen SH, Schousboe A, Hansen HS. 2000.
Determination of the phospholipid precursor of anandamide
and other N-acylethanolamine phospholipids before and
after sodium azide-induced toxicity in cultured neocortical
neurons. J Neurochem 75:861871.
Harrison KA, Clay KL, Murphy RC. 1999. Negative ion electrospray and tandem mass spectrometric analysis of platelet
activating factor (PAF) (1-hexadecyl-2-acetyl-glycerophosphocholine). J Mass Spectrom 34:330335.
Harrison KA, Davies SS, Marathe GK, McIntyre T, Prescott S,
Reddy KM, Falck JR, Murphy RC. 2000. Analysis of
oxidized glycerophosphocholine lipids using electrospray
ionization mass spectrometry and microderivatization
techniques. J Mass Spectrom 35:224236.
Hartvigsen K, Ravandi A, Bukhave K, Holmer G, Kuksis A.
2001. Regiospecific analysis of neutral ether lipids by liquid
chromatography/electrospray ionization/single quadrupole

48

SHOTGUN LIPIDOMICS BY ESI/MS

&

Author Proof

mass spectrometry: Validation with synthetic compounds.


J Mass Spectrom 36:11161124.
Hazen SL, Hall CR, Ford DA, Gross RW. 1993. Isolation of a
human myocardial cytosolic phospholipase A2 isoform. Fast
atom bombardment mass spectroscopic and reverse-phase
high pressure liquid chromatography identification of
choline and ethanolamine glycerophospholipid substrates.
J Clin Invest 91:25132522.
Hoff HF, ONeil J, Wu Z, Hoppe G, Salomon RL. 2003.
Phospholipid hydroxyalkenals: Biological and chemical
properties of specific oxidized lipids present in atherosclerotic lesions. Arteriocler Thromb Vasc Biol 23:275
282.
Hoischen C, Ihn W, Gura K, Gumpert J. 1997. Structural characterization of molecular phospholipid species in cytoplasmic
membranes of the cell wall-less Streptomyces hygroscopicus L form by use of electrospray ionization coupled
with collision-induced dissociation mass spectrometry.
J Bacteriol 179:34373442.
Horrocks LA, Sharma M. 1982. Plasmalogens and O-alkyl
glycerophospholipids. In: Hawthorne JN, Ansell GB,
editors. Phospholipids. Amsterdam, The Netherlands:
Elsevier Biomedical Press. pp 5193.
Hsu F-F, Bohrer A, Turk J. 1998a. Electrospray ionization
tandem mass spectrometric analysis of sulfatide. Determination of fragmentation patterns and characterization of
molecular species expressed in brain and in pancreatic islets.
Biochim Biophys Acta 1392:202216.
Hsu F-F, Bohrer A, Turk J. 1998b. Formation of lithiated
adducts of glycerophosphocholine lipids facilitates their
identification by electrospray ionization tandem mass spectrometry. J Am Soc Mass Spectrom 9:516526.
Hsu F-F, Turk J. 1999. Structural characterization of triacylglycerols as lithiated adducts by electrospray ionization mass
spectrometry using low-energy collisionally activated dissociation on a triple stage quadrupole instrument. J Am Soc
Mass Spectrom 10:587599.
Hsu F-F, Turk J. 2000. Characterization of phosphatidylinositol,
phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem
mass spectrometry: A mechanistic study. J Am Soc Mass
Spectrom 11:986999.
Hsu F-F, Turk J. 2001. Structural determination of glycosphingolipids as lithiated adducts by electrospray ionization
mass spectrometry using low-energy collisional-activated
dissociation on a triple stage quadrupole instrument. J Am
Soc Mass Spectrom 12:6179.
Hsu F-F, Turk J. 2002. Characterization of ceramides by low
energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization. J Am
Soc Mass Spectrom 13:558570.
Hsu F-F, Bohrer A, Wohltmann M, Ramanadham S, Ma Z,
Yarasheski K, Turk J. 2000a. Electrospray ionization mass
spectrometric analyses of changes in tissue phospholipid
molecular species during the evolution of hyperlipidemia

and hyperglycemia in Zucker diabetic fatty rats. Lipids


35:839854.
Hsu F-F, Ma Z, Wohltmann M, Bohrer A, Nowatzke W,
Ramanadham S, Turk J. 2000b. Electrospray ionization/
mass spectrometric analyses of human promonocytic U937
cell glycerolipids and evidence that differentiation is
associated with membrane lipid composition changes that
facilitate phospholipase A2 activation. J Biol Chem 275:
1657916589.
Hsu F-F, Turk J, Thukkani AK, Messner MC, Wildsmith KR,
Ford DA. 2003. Characterization of alkylacyl, alk-1enylacyl and lyso subclasses of glycerophosphocholine by
tandem quadrupole mass spectrometry with electrospray
ionization. J Mass Spectrom 38:752763.
Hvattum E, Larsen A, Uran S, Michelsen PM, Skotland T. 1998.
Specific detection and quantification of palmitoyl-stearoylphosphatidylserine in human blood using normal-phase
liquid chromatography coupled with electrospray mass
spectrometry. J Chromatogr B 716:4756.
Igbavboa U, Hamilton J, Kim H-Y, Sun GY, Wood WG.
2002. A new role for apolipoprotein E: Modulating
transport of polyunsaturated phospholipid molecular species in synaptic plasma membranes. J Neurochem 80:255
261.
Ishida M, Hayakawa J, Imagawa M, Taguchi R. 2001. Microanalysis of phospholipids of mammalian cells by liquid
chromatography/electrospray ionization-mass spectrometry. J Mass Spectrom Soc Jpn 49:150160.
Ishizuka I, Yamakawa T. 1985. Glycoglycerolipids. In: Wiegandt
H, editor. Glycolipids. Amsterdam, The Netherlands:
Elsevier. pp 101197.
Ivanova PT, Cerda BA, Horn DM, Cohen JS, McLafferty FW,
Brown HA. 2001. Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3
mastocytoma cells during degranulation. Proc Natl Acad Sci
USA 98:71527157.
Johnson AW, Mills K, Clayton PT. 1996. The use of automated
electrospray ionization tandem MS for the diagnosis of
inborn errors of metabolism from dried blood spots.
Biochem Soc Trans 24:932938.
Kakela R, Somerharju P, Tyynela J. 2003. Analysis of
phospholipid molecular species in brains from patients with
infantile and juvenile neuronal-ceroid lipofuscinosis using
liquid chromatography-electrospray ionization mass spectrometry. J Neurochem 84:10511065.
Kalderon B, Sheena V, Shachrur S, Hertz R, Bar-Tana J. 2002.
Modulation by nutrients and drugs of liver acyl-CoAs
analyzed by mass spectrometry. J Lipid Res 43:11251132.
Kayganich KA, Murphy RC. 1992. Fast atom bombardment tandem mass spectrometric identification of diacyl,
alkylacyl, and alk-1-enylacyl molecular species of glycerophosphoethanolamine in human polymorphonuclear leukocytes. Anal Chem 64:29652971.
Kerwin JL, Tuininga AR, Ericsson LH. 1994. Identification
of molecular species of glycerophospholipids and

49

&

HAN AND GROSS

Author Proof

sphingomyelin using electrospray mass spectrometry. J


Lipid Res 35:11021114.
Kevala JH, Kim H-Y. 2001. Determination of substrate
preference in phosphatidylserine decarboxylation by liquid
chromatography-electrospray ionization mass spectrometry. Anal Biochem 292:130138.
Khan WA, Blobe GC, Hannun YA. 1995. Arachidonic acid and
free fatty acids as second messengers and the role of protein
kinase C. Cell Signal 7:171184.
Khaselev N, Murphy RC. 2000. Electrospray ionization mass
spectrometry of lysoglycerophosphocholine lipid subclasses. J Am Soc Mass Spectrom 11:283291.
Kim H-Y, Akbar M, Kim K-Y. 2001. Inhibition of neuronal
apoptosis by polyunsaturated fatty acids. J Mol Neurosci
16:223227.
Kim H-Y, Wang TCL, Ma Y-C. 1994. Liquid chromatography/
mass spectrometry of phospholipids using electrospray
ionization. Anal Chem 66:39773982.
Kimelberg HK. 1985. Membrane fluidity and lipid composition.
NATO ASI Series, Series A: Life Sci 71:261276.
Koivusalo M, Haimi P, Heikinheimo L, Kostiainen R,
Somerharju P. 2001. Quantitative determination of phospholipid compositions by ESI-MS: Effects of acyl chain
length, unsaturation, and lipid concentration on instrument
response. J Lipid Res 42:663672.
Kolesnick RN, Goni FM, Alonso A. 2000. Compartmentalization
of ceramide signaling: Physical foundations and biological
effects. J Cell Physiol 184:285300.
Kolesnick RN, Kronke M. 1998. Regulation of ceramide
production and apoptosis. Annu Rev Physiol 60:643
665.
Kostiainen R, Tuominen J, Kokkonen J, Heikinheimo L,
Somerharju P. 1998. Determination of phospholipid
composition of cultured cells by electrospray-tandem
mass spectrometry. Adv Mass Spectrom 14:C052140/1
C052140/7.
Lee SH, Williams MV, DuBois RN, Blair IA. 2003. Targeted
lipidomics using electron capture atmospheric pressure
chemical ionization mass spectrometry. Rapid Commun
Mass Spectrom 17:21682176.
Lehmann WD, Koester M, Erben G, Keppler D. 1997.
Characterization and quantification of rat bile phosphatidylcholine by electrospray-tandem mass spectrometry. Anal
Biochem 246:102110.
Lennartz MR. 1999. Phospholipases and phagocytosis: The role
of phospholipid-derived second messengers in phagocytosis. Int J Biochem Cell Biol 31:415430.
Liebisch G, Drobnik W, Reil M, Trumbach B, Arnecke R,
Olgemoller B, Roscher A, Schmitz G. 1999. Quantitative
measurement of different ceramide species from crude
cellular extracts by electrospray ionization tandem mass
spectrometry (ESI-MS/MS). J Lipid Res 40:15391546.
Liebisch G, Drobnik W, Lieser B, Schmitz G. 2002. Highthroughput quantification of lysophosphatidylcholine by
electrospray ionization tandem mass spectrometry. Clic
Chem 48:22172224.

Lieser B, Liebisch G, Drobnik W, Schmitz G. 2003. Quantification of sphingosine and sphinganine from crude lipid
extracts by HPLC electrospray ionization tandem mass
spectrometry. J Lipid Res 44:22092216.
Listenberger L, Han X, Lewis SE, Cases S, Farese RV Jr, Ory DS,
Schaffer JE. 2003. Unsaturated fatty acids prevent lipotoxicity through induction of triglyceride accumulation. Proc
Natl Acad Sci USA 100:30773082.
MahleyQ4 RW. 1988. Apolipoprotein E: Cholesterol transport
protein with expanding role in cell biology. Science
240:622630.
Mancuso DJ, Abendschein DR, Jenkins CM, Han X, Saffitz JE,
Schuessler RB, Gross RW. 2003. Cardiac ischemia activates
calcium-independent phospholipase A2b, precipitating ventricular tachyarrhythmias in transgenic mice: Rescue of the
lethal electrophysiologic phenotype by mechanism-based
inhibition. J Biol Chem 278:2223122236.
Mano N, Oda Y, Yamada K, Asakawa N, Katayama K. 1997.
Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray
ionization tandem mass spectrometry. Anal Biochem 244:
291300.
Markesbery WR. 1997. Oxidative stress hypothesis in Alzheimers disease. Free Radic Biol Med 23:134147.
McClellan JE, Quarmby ST, Yost RA. 2002. Parent and neutral
loss monitoring on a quadrupole ion trap mass spectrometer: Screening of acylcarnitines in complex mixtures.
Anal Chem 74:57995806.
McCluerQ5 RH, Ullman MD, Jungalwala FB. 1986. HPLC of
glycosphingolipids and phospholipids. Adv Chromatogr
25:309353.
Mclafferty FW, Turecek F. 1993. Interpretation of mass spectra.
Sausalito, California: University Science Books.
McLafferty FW, Senko MW, Little DP, Wood TD, OConnor PB,
Speir JP, Chorush RA, Kelleher NL. 1995. Electrospray ionization and fourier-transform mass spectrometry
in biomedical research. Adv Mass Spectrom 13:115
128.
Meade EA, Jones DA, Zimmerman GA, McIntyre TM,
Prescott SM. 1996. Prostaglandins and related compounds.
Lipid messengers with many actions. Handbook Lipid Res
8:285305.
MeisenQ6 I, Peter-Katalinic J, Muething J. 2003. Discrimination
of neolacto-series gangliosides with a2-3- and a2-6-linked
N-acetylneuraminic acid by nanoelectrospray ionization
low-energy collision-induced dissociation tandem quadrupole TOF MS. Anal Chem 75:57195725.
Merrill AH Jr. 2002. De novo sphingolipid biosynthesis: A
necessary, but dangerous, pathway. J Biol Chem 277:
2584325846.
Merrill AH Jr, Sweeley CC. 1996. Sphingolipids: Metabolism
and cell signalling. In: Vance DE, Vance J, editors.
Biochemistry of lipids, lipoproteins and membranes.
Amsterdam, The Netherlands: Elsevier. pp 309339.
Moeder M, Kiessling A, Loester H, Brueggemann L. 2003. The
pattern of urinary acylcarnitines determined by electrospray

50

SHOTGUN LIPIDOMICS BY ESI/MS

&

Author Proof

mass spectrometry: A new tool in the diagnosis of diabetes


mellitus. Anal Bioanal Chem 375:200210.
Moolenaar WH. 1995. Lysophosphatidic acid signalling. Curr
Op Cell Biol 7:203210.
Murphy RC. 2001. Free-radical-induced oxidation of arachidonoyl plasmalogen phospholipids: Antioxidant mechanism
and precursor pathway for bioactive eicosanoids. Chem Res
Toxicol 14:463472.
Murphy RC, Fiedler J, Hevko J. 2001. Analysis of nonvolatile lipids by mass spectrometry. Chem Rev 101:479
526.
Murthy M, Hamilton J, Greiner RS, Moriguchi T, Salem N Jr,
Kim H-Y. 2002. Differential effects of n-3 fatty acid
deficiency on phospholipid molecular species composition
in the rat hippocampus. J Lipid Res 43:611617.
Newberry NP, Xie Y, Kennedy S, Han X, Buhman KK, Luo J,
Gross RW, Davidson NO. 2003. Decreased hepatic triglyceride accumulation and altered fatty acid uptake in mice
with deletion of the liver fatty acid binding protein gene.
J Biol Chem 278:5166451672.
Pak JH, Han X, Gross RW. 1992. Differential molecular
dynamics and transmembrane fluidity gradients in canine
myocardial sarcolemma and sarcoplasmic reticulum. Chem
Phys Lipids 61:111119.
Paola MD, Cocco T, Lorusso M. 2000. Ceramide interaction with
the respiratory chain of heart mitochondria. Biochemistry
39:66606668.
Pike LJ. 2003. Lipid rafts: Bringing order to chaos. J Lipid Res
44:655667.
Pike LJ, Han X, Chung K-N, Gross RW. 2002. Lipid rafts are
enriched in arachidonic acid and plasmenylethanolamine
and their composition is independent of caveolin-1 expression: A quantitative electrospray ionization/mass spectrometric analysis. Biochemistry 41:20752088.
PlumpQ7 AS, Breslow JL. 1995. Apolipoprotein E and the
apolipoprotein E-deficient mouse. Annu Rev Nutr 15:495
518.
Podrez EA, Poliakov E, Shen Z, Zhang R, Deng Y, Sun M, Finton
PJ, Shan L, Febbraio M, Hajjar DP, Silverstein RL, Hoff HF,
Salomon RG, Hazen SL. 2002. A novel family of
atherogenic oxidized phospholipids promotes macrophage
foam cell formation via the scavenger receptor CD36 and is
enriched in atherosclerotic lesions. J Biol Chem 277:
3851738523.
Postle AD, Mander A, Reid KBM, Wang J-Y, Wright SM,
Moustaki M, Warner JO. 1999. Deficient hydrophilic lung
surfactant proteins A and D with normal surfactant phospholipid molecular species in cystic fibrosis. Am J Respir
Cell Mol Biol 20:9098.
Presti FT. 1985. Role of cholesterol in regulating membrane
fluidity. Membr Fluid Biol 4:97146.
Pulfer M, Murphy RC. 2003. Electrospray mass spectrometry of
phospholipids. Mass Spec Rev 22:332364.
Qiu D-F, Games MPL, Xiao X-Y, Games DE, Walton TJ.
2000. Characterization of membrane phospholipids and
glycolipids from a halophilic archaebacterium by high-

performance liquid chromatography/electrospray mass


spectrometry. Rapid Commun Mass Spectrom 14:1586
1591.
Raith K, Neubert RHH. 1998. Structural studies on ceramides by
electrospray tandem mass spectrometry. Rapid Commun
Mass Spectrom 12:935938.
Raith K, Neubert RHH. 2000. Liquid chromatography-electrospray mass spectrometry and tandem mass spectrometry of
ceramides. Anal Chim Acta 403:295303.
Ramanadham S, Hsu F-F, Bohrer A, Nowatzke W, Ma Z, Turk J.
1998. Electrospray ionization mass spectrometric analyses
of phospholipids from rat and human pancreatic islets and
subcellular membranes: Comparison to other tissues and
implications for membrane fusion in insulin exocytosis.
Biochemistry 37:45534567.
Rashed MS, Ozand PT, Harrison ME, Watkins PJF, Evans S.
1994. Electrospray tandem mass spectrometry in the diagnosis of organic acidemias. Rapid Commun Mass Spectrom
8:129133.
Ravandi A, Kuksis A, Marai L, Myher JJ, Steiner G, Lewisa G,
Kamido H. 1996. Isolation and identification of glycated
aminophospholipids from red cells and plasma of diabetic
blood. FEBS Lett 381:7781.
RayleighQ8 JW. 1882. XXXXX. S Philos Mag 14:184.
Rietveld A, Neutz S, Simons K, Eaton S. 1999. Association of
sterol- and glycosylphosphatidylinositol-linked proteins
with Drosophila raft lipid microdomains. J Biol Chem
274:20492054Q9.
Roberts LJ II. 2002. Lipids as regulators of cell function. Cell
Mol Life Sci 59:727728.
Rock CO, Jackowski S, Cronan JE Jr. 1996. Lipid metabolism in
prokaryotes. In: Vance DE, Vance J, editors. Biochemistry
of lipids, lipoproteins and membranes. Amsterdam, The
Netherlands: Elsevier. pp 3574.
Rodgers RP, Blumer EN, Emmett MR, Marshall AG. 2000.
Efficacy of bacterial bioremediation: Demonstration of
complete incorporation of hydrocarbons into membrane
phospholipids from rhodococcus hydrocarbon degrading
bacteria by electrospray ionization fourier transform ion
cyclotron resonance mass spectrometry. Environ Sci
Technol 34:535540.
Sandhoff R, Brugger B, Jeckel D, Lehmann WD, Wieland FT.
1999. Determination of cholesterol at the low picomole level
by nano-electrospray ionization tandem mass spectrometry.
J Lipid Res 40:126132.
Scherrer LA, Gross RW. 1989. Subcellular distribution, molecular dynamics and catabolism of plasmalogens in myocardium. Mol Cell Biochem 88:97105.
Schneiter R, Brugger B, Sandhoff R, Zellnig G, Leber A, Lampl
M, Athenstaedt K, Hrastnik C, Eder S, Daum G, Paltauf F,
Wieland FT, Kohlwein SD. 1999. Electrospray ionization
tandem mass spectrometry (ESI-MS/MS) analysis of the
lipid molecular species composition of yeast subcellular
membranes reveals acyl chain-based sorting/remodeling of
distinct molecular species en route to the plasma membrane.
J Cell Biol 146:741754.

51

&

HAN AND GROSS

Author Proof

Schuck S, Honsho M, Ekroos K, Shevchenko A, Simons K. 2003.


Resistance of cell membranes to different detergents. Proc
Natl Acad Sci USA 100:57955800.
Seal JR, Havrilla CM, Porter NA, Hachey DL. 2003. Analysis of
unsaturated compounds by Ag coordination ionspray mass
spectrometry: Studies of the formation of the Ag/lipid
complex. J Am Soc Mass Spectrom 14:872880.
Smith PBW, Snyder AP, Harden CS. 1995. Characterization of
bacterial phospholipids by electrospray ionization tandem
mass spectrometry. Anal Chem 67:18241830.
Smith RD, Loo JA, Edmonds CG, Barinaga CJ, Udseth HR. 1990.
New developments in biochemical mass spectrometry:
Electrospray ionization. Anal Chem 62:882899.
Spector AA, Yorek MA. 1985. Membrane lipid composition and
cellular function. J Lipid Res 26:10151035.
Spickett CM. 2003. Analysis of phospholipid oxidation by
electrospray mass spectrometry. Crit Rev Oxidative Stress
Aging 1:393409.
Stanley J. 2001. Lipoproteins. 3. Why do lipoproteins promote
atherosclerosis? Lipid Technol 13:8990.
Strittmatter WJ, Roses AD. 1996. Apolipoprotein E and
Alzheimer disease. Annu Rev Neurosci 19:5377.
Sugita M, Itonori S, Inagaki F, Hori T. 1989. Characterization of
two glucuronic acid-containing glycosphingolipids in larvae
of the green-bottle fly, Lucilia caesar. J Biol Chem 264:
1502815033.
Sullards MC. 2000. Analysis of sphingomyelin, glucosylceramide, ceramide, sphingosine, and sphingosine 1-phosphate
by tandem mass spectrometry. Methods Enzymol 312:
3245.
Sullards MC, Wang E, Peng Q, Merrill AH Jr. 2003. Metabolomic
profiling of sphingolipids in human glioma cell lines by
liquid chromatography tandem mass spectrometry. Cell Mol
Biol 49:789797.
Sweetman G, Trinei M, Modha J, Kusel J, Freestone P, Fishov I,
Joseleau-Petit D, Redman C, Farmer P, Norris V. 1996.
Electrospray ionization mass spectrometric analysis of phospholipids of Escherichia coli. Mol Microbiol 20:233234.
Taguchi R, Hayakawa J, Takeuchi Y, Ishida M. 2000. Twodimensional analysis of phospholipids by capillary liquid
chromatography/electrospray ionization mass spectrometry. J Mass Spectrom 35:953966.
Thomas RL Jr, Matsko CM, Lotze MT, Amoscato AA. 1999.
Mass spectrometric identification of increased C16 ceramide levels during apoptosis. J Biol Chem 274:30580
30588.
Thukkani AK, Albert CJ, Wildsmith KR, Messner MC,
Martinson BD, Hsu F-F, Ford DA. 2003. Myeloperoxidase-derived reactive chlorinating species from human
monocytes target plasmalogens in low density lipoprotein.
J Biol Chem 278:3636536372.
Unger RH. 2002. Lipotoxic diseases. Ann Rev Med 53:319336.
Unger RH, Orci L. 2001. Diseases of liporegulation: New
perspective on obesity and related disorders. FASEB J
15:312321.

Vreken P, van Lint AEM, Bootsma AH, Overmars H,


Wanders RJA, van Gennip AH. 1999. Quantitative plasma
acylcarnitine analysis using electrospray tandem mass
spectrometry for the diagnosis of organic acidaemias and
fatty acid oxidation defects. J Inherit Metab Dis 22:302
306.
Watson AD, Subbanagounder G, Welsbie DS, Faull KF,
Navab M, Jung ME, Fogelman AM, Berliner JA. 1999.
Structural identification of a novel pro-inflammatory
epoxyisoprostane phospholipid in mildly oxidized low
density lipoprotein. J Biol Chem 274:2478724798.
Weintraub ST, Pinckard RN, Hail M. 1991. Electrospray
ionization for analysis of platelet-activating factor. Rapid
Commun Mass Spectrom 5:309911.
Weisgraber KH, Mahley RW. 1996. Human apolipoprotein E:
The Alzheimers disease connection. FASEB J 10:1485
1494.
Welti R, Wang X, Williams TD. 2003. Electrospray ionization
tandem mass spectrometry scan modes for plant chloroplast
lipids. Anal Biochem 314:149152.
Welti R, Wang X. 2004. Lipid species profiling: A high
throughput approach to identify lipid compositional changes
and determine the function of genes involved in lipid metabolism and signaling. Curr Op Plant Biol. In pressQ10.
Welti R, Li W, Li M, Sang Y, Biesiada H, Zhou H-E, Rajashekar
CB, Williams TD, Wang X. 2002. Profiling membrane lipids
in plant stress responses. Role of phospholipase Da in
freezing-induced lipid changes in Arabidopsis. J Biol Chem
277:3199432002.
Wenk MR, Lucast L, Di Paolo G, Romanelli AJ, Suchy SF,
Nussbaum RL, Cline GW, Shulman GI, McMurray W, De
Camilli P. 2003. Phosphoinositide profiling in complex lipid
mixtures using electrospray ionization mass spectrometry.
Nat Biotechol 21:813817.
Williams SD, Hsu F-F, Ford DA. 2000. Electrospray ionization mass spectrometry analyses of nuclear membrane
phospholipid loss after reperfusion of ischemic myocardium. J Lipid Res 41:15851595.
Xiao Y, Chen Y, Kennedy AW, Belinson J, Xu Y. 2000.
Evaluation of plasma lysophospholipids for diagnostic
significance using electrospray ionization mass spectrometry (ESI-MS) analyses. Ann NY Acad Sci 905:242
259.
Xiao Y, Schwartz B, Washington M, Kennedy A, Webster K,
Belinson J, Xu Y. 2001. Electrospray ionization mass
spectrometry analysis of lysophospholipids in human ascitic
fluids: Comparison of the lysophospholipid contents in
malignant vs. nonmalignant ascitic fluids. Anal Biochem
290:302313.
Xie Y, Gibbs TC, Mukhin YV, Meier KE. 2002. Role for 18:1
lysophosphatidic acid as an autocrine mediator in prostate
cancer cells. J Biol Chem 277:3251632526.
Yang J, Han X, Gross RW. 2003. Identification of hepatic
peroxisomal phospholipase A2 and characterization of
arachidonic acid-containing choline glycerophospholipids
in hepatic peroxisomes. FEBS Lett 546:247250.

52

SHOTGUN LIPIDOMICS BY ESI/MS

&

Author Proof

Yoon H-R, Kim H, Cho S-H. 2003. Quantitative analysis of acyllysophosphatidic acid in plasma using negative ionization
tandem mass spectrometry. J Chromatog B 788:8592.
Zacarias A, Bolanowski D, Bhatnagar A. 2002. Comparative
measurements of multicomponent phospholipid mixtures by

electrospray mass spectroscopy: Relating ion intensity to


concentration. Anal Biochem 308:152159.
Zoeller RA, Morand OH, Raetz CRH. 1988. A possible role for
plasmalogens in protecting animal cells against photosensitized killing. J Biol Chem 263:1159011596.

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