Beruflich Dokumente
Kultur Dokumente
Author Proof
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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
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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MAS-490
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Author Proof
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VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
I. INTRODUCTION
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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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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
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(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.
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
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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.
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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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.
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C. Metabolites
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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
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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.
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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.
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
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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.
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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!!!
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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.
??
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
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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.
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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.
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1. Lipid Extraction
2. Lipid Concentration
3. Internal Standards
PI internal std
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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.)
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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.)
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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.
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!!!
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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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.
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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.)
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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.
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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.)
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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
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.
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
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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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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.
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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Author Proof
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
&
Author Proof
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.)
37
&
Author Proof
&
Author Proof
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.)
39
&
Author Proof
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).
40
&
Author Proof
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.)
41
&
Author Proof
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.)
42
&
Author Proof
A
E. Alterations in Phospholipids in Mast
Cells During Degranulation
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.).
43
&
Author Proof
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
&
Author Proof
IX. ABBREVIATIONS
AA
AD
DAG
ESI
FAB
FTMS
GalC
m:n
VIII. CONCLUSION
ACKNOWLEDGMENTS
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
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