BR-9976A

From tissues to targets.

PROTEOMELAB IGY TM

PROTEOME PARTITIONING SOLUTIONS

 Biomarker discovery is like panning for gold . . .
Finding the nuggets gets easier when there is less sand. Within biofluid proteomes such as plasma
and serum, that “sand” is comprised of well-characterized proteins. Identifying the potentially
golden biomarkers in the presence of these highly abundant proteins has proven to be a serious
challenge to current analytical techniques.

Typical Protein Abundances in Human Plasma, Log10 Concentration in pg/ml

In serum and plasma, the
dynamic range of proteins 0 1 2 3 4 5 6 7 8 9 10 11 12
Hemoglobin
spans over 10 orders of Albumin
magnitude, far greater than IgG Total
Transferrin
the measurement capability Fibrinogen
IgA Total
of current technologies. α2-Macroglobulin
Additionally, potential IgM Total
α1-Antitrypsin
biomarkers are entirely C3 Complement
Haptoglobin
masked by the overwhelming Apolipoprotein A-I
abundance of relatively Apolipoprotein B
α1-acid Glycoprotein
few proteins. Lipoprotein (a)
Factor H
Ceruloplasmin
C4 Complement
Complement Factor B
Prealbumin
C9 Complement
C1q Complement
C8 Complement
C5 Complement
Plasminogen
IgD
C1 Inhibitor
C6 Complement
C7 Complement
Complement Factor I
iC3b
Retinol Binding Protein
Thyroxin Binding Globulin
C2 Complement Protein
Thrombus Precursor Protein
C-reactive Protein
Bb Fragment
C3a Complement Protein
Ferritin
Rantes
SC5b-9 Complex
Myoglobin
Thyroglobulin
TPA
C5a Complement
Neuron-Specific Enclase
C-peptide
α-Fetoprotein
TNF-Binding Proteins
Prostate-Specific Antigen
Prostatic Acid Phosphatase
CEA
Myelin Basic Protein
Troponin I
Interleukin-1ra
MIP-1 beta
Typical Range Abundances Troponin T
Interleukin-8
Courtesy of MIP-1 alpha
Plasma Proteome Institute Tissue Factor
GCSF
Interferon Alpha
Interleukin-2 Normally Not Found in Plasma
The human plasma Interleukin-4
Classical Plasma Proteins
proteome: History, character, TNF-Alpha
Interferon Gamma
and diagnostic prospects. Interleukin-1 Beta Tissue Leakage

Anderson, N.L. and Interleukin-12

Interleukin-10 Interleukins & Cytokines
Anderson, N.G., Molecular Interleukin-5
Other
Interleukin-6
and Cellular Proteomics,
1.11, 845-867 (2002).
 Transferrin Fibrinogen
12 Proteins comprise up to 96%
IgG Total
IgA
α2-Macroglobulin
of the protein mass in plasma.
IgM
α1-Antitrypsin
Haptoglobin
α1-acid Glycoprotein
Apolipoprotein A-I
Apolipoprotein A-II
POOL CONTAINING
LOW ABUNDANT
PROTEINS

Proteome partitioning.
Protein depletion has been used for some years to
remove most of the albumin and/or IgG from biofluids such as
Albumin plasma and serum prior to analysis, but it is clear that this alone
is insufficient to enable progress to be made in biomarker discovery.
The presence of highly abundant proteins significantly complicates the discovery
process by masking the presence and limiting the detection of low abundance species.
ProteomeLab IgY partitioning addresses this issue by reversibly capturing 12 of the more abundant
proteins from human biofluids such as plasma and serum, yielding an enriched pool of low abundance
proteins for further study. The captured proteins can also be easily recovered for investigation if required
- hence the term partitioning rather then depletion.

IgY-12 will selectively partition the 12 highly abundant proteins highlighted in the above illustration.
The partitioned fractions can be taken to the next stage of the discovery process, such as multi-dimensional
fractionation using the ProteomeLab PF 2D system or profiling using 2D PAGE.

MW MW MW
A2MG
rs
arke

A1AT
IgM TRFE
MW M

IgA ALBU
A1AT
FIB-B
FIB-G IgG-HC
HPT
A1AG

APA1 IgG-LC

HPT

pH 3 10 pH 3 10 pH 3 10

Unfractionated Human Serum 12 Bound Abundant Proteins Enriched Low Abundance Proteins
(shown here with similar protein mass load)

Analysis of partitioned human serum by 2D PAGE in this 3D rendering illustrates the mountains of highly abundant proteins that
can be separated and the less abundant proteins in the enriched sample that then become apparent.
 The ProteomeLab solution.
The ProteomeLab IgY Partitioning system coupled with the ProteomeLab PF 2D Protein Fractionation
system, facilitates significant advancement in the search for biomarkers. The IgY-12 Proteome Parti-
tioning Chemistry is optimized to capture 12 highly abundant protein species from primate samples,
whereas IgY-R7 is optimized to capture the 7 highly abundant protein species from rodent samples.
The ProteomeLab PF 2D further simplifies the proteome through fractionation into liquid phase.

The ProteomeLab PF 2D fractionates a biofluid proteome into hundreds of liquid phase fractions and generates differential displays
for the comparison of these proteomes. As total mass loads of up to 5 mg of protein may be fractionated per analysis, proteins of
medium to low abundance will be enriched while more highly abundant proteins that typically mask biomarker discovery are placed
into discrete fractions. By coupling the enrichment power of the IgY-12 proteome partitioning with the fractionation power of the
ProteomeLab PF 2D you will vastly enhance the discovery and validation of biomarkers and drug targets from biofluids such as
plasma and serum.

The figures below illustrate data resulting from coupling these two techniques.

Whole plasma Plasma partitioned by IgY-12

These two protein maps are visualizations from the ProteomeLab PF 2D fractionation generated from a 3 mg
load of whole human plasma and an equal total protein mass load of enriched low to medium abundant
proteins from IgY-12 partitioned plasma. These maps illustrate that the highly abundant proteins are selectively
partitioned while the low to medium abundant proteins are enriched.

<4.45 4.45-4.75 4.76-5.06 5.07-5.37 5.38-5.68 5.69-5.99 6.0-6.3 6.31-6.61 6.62-6.92 6.93-7.23

The differential display map generated by the ProteomeLab PF 2D software allows you to visualize the differences that exist between
whole plasma versus the enriched/partitioned plasma. The red bands illustrate proteins that predominate in whole plasma – which
in this case are effectively the 12 major proteins being partitioned out. The green bands represent proteins that predominate in the
partitioned sample representing enrichment of the low to medium abundance proteins. All of these proteins are collected as liquid
phase fractions ready for characterization.
Chromatogram overlays from related pH regions of the differential display map highlight significant numbers of low to medium
abundance proteins that are clearly enriched (green trace) relative to those that predominated (red trace) in the unpartitioned plasma.

Conversely, the bands highlighted in the red trace are those highly abundant proteins that are predominant in whole plasma and
have been removed from the partitioned plasma. This is illustrated in chromatogram overlays from pH fractions 5.69 – 5.99 where
both human serum albumin and apolipoprotein A-I are predominant (red trace), and in chromatogram overlays from pH fraction
6.62 – 6.92 where transferrin is highly abundant (red trace).

Overlay of chromatograms from pH fraction

less than 4.45.

Overylay of chromatograms
from pH fraction 4.45 to 4.75.

Overlay of chromatograms from pH

fraction 5.69 – 5.99 where human
serum albumin (16.5 min) and
apolipoprotein A-I (19 min) are highly
abundant (red trace) in whole plasma,
while they are absent in the
partitioned plasma (green trace).

Overlay of chromatograms from pH fraction

6.62 – 6.92 where transferrin is highly abun-
dant in whole plasma (16 min red trace),
whereas its removal is highlighted from the
partitioned plasma (green trace).
 Why IgY?
IgY antibodies are produced by injecting an avian species (chicken) with highly purified mammalian
protein antigens. IgY chemistries offer broader antigen-binding host range and cleaner capture than
IgG capture methods because of the evolutionary distance between chicken and mammals.

Broader antigen-binding host range.

Due to the phylogenetic difference,
mammalian proteins are often more
immunogenic in birds than in mammals,*
hence the broader antigen-binding host range.
ProteomeLab IgY Chemistry effectively
leverages this well-documented
evolutionary advantage.

*Gassman, M., Thoemmes, P., Weiser, T., and Multiple Protein Alignment
Huebscher, U. (1990) Efficient production of using Clustal X & TreeView
chicken egg yolk antibodies against a conserved
mammalian protein. FASEB J. 4, 2528-2532 Numbers represent % amino
acid similarity to HSA

Ag Ag Ag Ag

Contrary to IgG, IgY does not bind in a

non-specific manner to the proteins
IgY Fc
IgG listed. Combined with its broader
host range, this is why IgYs are
the antibodies of choice for
Fc Binding proteome partitioning.
IgM or Fc Receptors
Rheumatoid Factor
Complement Factors
X Protein A or G
NO Binding Sample

Cleaner capture. Partitioning Efficiency

% Method of
Protein Removal Detection
As the polyclonal IgYs are avian, the Fc region of the Albumin >99.5 ELISA
antibody does not bind mammalian complement factors, IgG Total >99.5 W. Blot
Rheumatoid Factor, IgM, Fc receptor and Protein A or Transferrin >99.5 W. Blot
G, a known issue related to capture with mammalian Haptaglobin 92.5 W. Blot
antibodies. Coupled with high specificity across multiple α1-Antitrypsin >95 W. Blot
epitopes, you get very specific binding across species while
α2-Macroglobulin >99.5 W. Blot
achieving very clean capture with little background.
IgA Total >99.5 W. Blot
The SDS-PAGE highlights whole serum run in lane #1,
serum with just albumin partitioned out by ProteomeLab IgM Total 90-95 ELISA
IgY-HSA SC chemistry in lane #2, and the isolated/ α1-acid Glycoprotein 92-95 W. Blot
partitioned albumin captured cleanly in lane #3. The Apolipoprotein A-I >99.5 W. Blot
partitioning efficiency of the reagents from human Apolipoprotein A-II >99.5 W. Blot
plasma is highlighted in the adjoining table. To further Fibrinogen >99.5 W. Blot
improve capture, Beckman Coulter offers you both
primate optimized 12-plex, and rodent optimized 7-plex
affinity solutions.
1 2 3
 Why partitioning?
Rather than employing a depletion strategy, proteome partitioning
focuses on both the affinity capture of highly abundant proteins as
well as the enrichment of the flow-through material.

50 mg Plasma or Serum

Affinity Capture Whether you are fractionating

IgY-12 or IgY-R7 or profiling biofluid proteomes,
Proteome Partitioning the presence of highly abundant
proteins will mask the identity
5 cycles of many of the medium to
lower abundance species.

Now you can greatly enhance

Captured Highly Isolate Individual Proteins
Abundant Proteins IgY Spin Columns
Protein Determination
ProteomeLab DU 800
ProteomeLab PF 2D Proteome Fractionation
Proteome Map
Immunoassay Mass Spectrometry
Protein Identification
the detectability and identifi-
Flow-through cation of less abundant
Enriched Proteins proteins through the selective
partitioning of many of the
highly abundant species. This
Concentrate Fractions
process serves to both enrich
ProteomeLab SP System
the medium to low abundance
proteins, while removing the
deleterious masking effects
generated by the overlapping
peptide mass fingerprints from
highly abundant proteins. In
parallel, the captured highly
abundant proteins are collected
to allow for further analysis, as
these proteins may also include
Fractions a wealth of information. Either
way, all protein fractions are
preserved to allow you to
Other monitor all aspects of the
proteome.

Imagine taking the equivalent of 50 mg of plasma or serum protein

for a single fractionation experiment – this in fact is what you will be
doing by combining the capacity of the ProteomeLab IgY-12 LC10
column with the fractionation power of the ProteomeLab PF 2D
system. Five cycles from the IgY-12 affinity column will enrich
approximately 5 mg of protein from 50 mg of plasma or serum
protein – which the PF 2D system will fractionate into several
hundred fractions using pI in the first dimension and
hydrophobicity in the second dimension – bringing a whole
new layer of proteins into focus.
 ProteomeLab IgY chemistries.
The ProteomeLab IgY proteome partitioning chemistries are based on affinity columns using avian (IgY)
antigen interactions and optimized buffers for sample loading, washing, eluting and regenerating. The
selective immunoaffinity partitioning provides an enriched pool of lower abundant proteins.

The ProteomeLab IgY-12 proteome partitioning chemistries are specifically designed to remove twelve highly abundant proteins from
human/primate biological fluids such as serum, plasma, and cerebral spinal fluid (CSF). This technology enables removal of albumin,
total IgG, α1-antitrypsin, IgA, IgM, transferrin, haptoglobin, α1-acid glycoprotein (orosmucoid), α2-macroglobulin, HDL
(apolipoproteins A-I & A-II) and fibrinogen in a single step.

The ProteomeLab IgY-R7 proteome partitioning chemistries are specifically designed to remove seven highly abundant proteins from
rodent biological fluids such as serum, plasma, and cerebral spinal fluid (CSF). This technology enables removal of albumin, IgG, α1-
antitrypsin, IgM, transferrin, haptoglobin and fibrinogen, in a single step.

In both cases, the targeted highly abundant proteins are simultaneously partitioned by the immobilized specific IgYs when complex bio-
logical samples are passed through the column. Selective immunoaffinity partitioning provides an enriched pool of lower abundant pro-
teins for downstream proteomic analysis.

The format of which chemistry to use should be selected on the basis of:
- The volume of biological fluids needed to yield the target quantity of partitioned protein for subsequent analysis
- The sample throughput requirements of your lab

Chemistry formats.
The IgY spin column format is intended for more analytical scale analyses
and utilizes centrifugation as the force for affinity separation. The IgY SC
spin column format is available in IgY-12 and IgY-R7, as well as individual
partitioning solutions for HSA, BSA, RSA, total IgG, fibrinogen,
transferrin, and HDL. As two spin columns are provided with each kit,
you get double the throughput of other spin column solutions.

The IgY LC2 column format is a moderate capacity chemistry utilizing IgY
microbeads packed into a 2 ml column bed with liquid chro-
matography used as the force for affinity separation.
This column has a capacity of 25 µL human/
primate serum or plasma per LC column cycle,
while the IgY-R7 LC2 column kit has a capacity of 20 µL
mouse or 40 µL rat serum/plasma per LC column cycle.

The IgY LC10 column format is a high capacity chemistry

utilizing IgY microbeads packed into a 10 ml column
bed with liquid chromatography used as the force
for affinity separation. The IgY-R7 LC10
column has a capacity of 100 µL mouse or
200 µL rat serum/plasma per LC column cycle.

The above formats have been integrated into the ProteomeLab

IgY spin column and ProteomeLab high capacity solutions.
 ProteomeLab IgY spin column solution.
When your focus is more geared to analytical scale processing for 2D gel profiling, the ProteomeLab IgY proteome partitioning
solution is ideal. Our spin column solution is designed to process 10 µl of human or 15 µl of rat plasma/serum per cycle.

Combining the innovative IgY chemistry with

Beckman Coulter’s proven separation technologies
provides a convenient solution to simplify and
enrich a plasma, serum or CSF proteome.

The ProteomeLab IgY spin column solution

includes the ProteomeLab SP Sample Preparation
system, validated protocols in the form of standard
operating procedures and the IgY Spin Column
chemistry, configured as either IgY-12, which
partitions 12 highly abundant proteins from
human/primate biofluids or IgY-R7 which is
optimized to partition seven highly abundant
proteins from rodent biofluids.

The IgY-12 spin column solution is designed to

process 10 µL human/primate serum or plasma per
spin column cycle. The expected yield of a sample
partitioned of the highly abundant proteins is about
80 µg. The IgY-R7 SC spin column has a capacity
of 10 µL mouse or 15 µL rat serum/plasma per
spin column cycle. The expected yield of a sample
partitioned of the 7 highly abundant proteins is
about 150 – 250 µg.

The ProteomeLab SP Sample Preparation system includes the

necessary validated protocols, separation module, sample prep rotors
with device adapters to process serum or plasma from whole blood,
and to provide the separation force for the spin column affinity chemistry.
 ProteomeLab IgY high capacity solution.
Combining the innovative IgY chemistry with Beckman Coulter’s proven separation technologies
provides a convenient solution to simplify and enrich a plasma, serum or CSF proteome. Our high
capacity solution is designed to process 125 µl of human or 200 µl of rat plasma/serum per cycle.

Combining the innovative IgY chemistry with Beckman Coulter’s proven

separation technologies provides a convenient solution to simplify and enrich
a plasma, serum or CSF proteome. Our high capacity solution includes the
ProteomeLab PPS Proteome Partitioning System, the ProteomeLab SP
Sample Preparation system and high capacity IgY Chemistry, capable of
processing 125 µL of human and 200 µL of rat plasma/serum per cycle.

The ProteomeLab PPS system consists of several components that integrate

with the ProteomeLab PF 2D, providing an advanced pump, automated
buffer switching and large volume fraction collection to implement the
immunoaffinity-based proteome partitioning.

The IgY partitioning methods are

built into the software with the
whole process managed by the
ProteomeLab PF 2D
software suite.

The integrated 4-way buffer The system includes validated

selection valve ensures highly optimized protocols and methods to ensure
reproducible step elution in success in partitioning your serum/plasma proteome.
an automated manner.
The IgY LC10 chemistry provides the capacity to enrich
larger quantities of medium to low abundant proteins.
The ProtomeLab IgY-12 and IgY-R7 are capable of
processing 125 µL of human and 200 µL of rat PROTEOMELAB TM

plasma/serum per cycle respectively. In both cases, FROM TISSUES TO TARGETS.

pooling 3 to 5 cycles from the LC10 column will yield
ample material for ProteomeLab PF 2D fractionation –
enabling you in the detection and Identify
identification of more low
abundance proteins.
Isolate

Fractionate

Characterize

Evaluate

Diagnose

The ProteomeLab SP
sample preparation system
includes validated methods,
standard operating protocols,
the necessary sample prep rotors
and device adapters to isolate plasma or serum
from whole blood, to clean up your sample before
introducing it onto the affinity column, and for concentrating Together with the ProteomeLab PF 2D, the IgY high
both the flow-through and bound proteins which will initially capacity proteome partitioning solution vastly enhances
be eluted from the column as a dilute solution. discovery and validation of biomarkers.
 • Flow cytometry
• Automated fluorescence microscopy
• Microarray technology
• Cell viability analysis
• General purpose centrifugation
• High performance centrifugation
• Ultracentrifugation
• Centrifugal elutriation
• Automated liquid handling
• Immunoaffinity partitioning
• High performance centrifugation
• Ultracentrifugation
• Liquid chromatography
• Capillary electrophoresis/
Mass spectrometry
• Spectrophotometry
• Protein characterization in solution
• Automated protein crystallization
• Microarray technology
• Software solutions
• Automated immunoassay
• Flow cytometry
• Electrophoresis
• Immunochemistry
• Immunofixation
IgY partitioned samples can be further fractionated
on the ProteomeLab PF 2D (shown above) prior
to mass spectrometry or other characterization
techniques.
B2005-6709-DG-5K
ORDERING INFORMATION
Solutions
ProteomeLab IgY High Capacity Proteome Partitioning Solution
Includes: ProteomeLab SP Sample Preparation System, ProteomeLab PPS Proteome
Partitioning System and ProteomeLab IgY-12 or IgY-R7 LC10 Proteome Partitioning
Chemistry.
ProteomeLab IgY Spin Column Proteome Partitioning Solution
Includes: ProteomeLab SP Sample Preparation System & ProteomeLab IgY-12 or
IgY-R7 SC Proteome Partitioning Chemistry.
Replacement Chemistry
ProteomeLab IgY-12 LC10 Proteome Partitioning Kit A24355
ProteomeLab IgY-12 LC2 Proteome Partitioning Kit A24346
ProteomeLab IgY-12 SC Proteome Partitioning Kit A24618
ProteomeLab IgY-R7 LC10 Proteome Partitioning Kit A25408
ProteomeLab IgY-R7 LC2 Proteome Partitioning Kit A25404
ProteomeLab IgY-R7 SC Proteome Partitioning Kit A25626
ProteomeLab IgY-HSA SC Proteome Partitioning Kit A25521
ProteomeLab IgY-BSA SC Proteome Partitioning Kit A25526
ProteomeLab IgY-RSA SC Proteome Partitioning Kit A25527
ProteomeLab IgY-IgG-Fc SC Proteome Partitioning Kit A25522
ProteomeLab IgY-Fibrinogen SC Proteome Partitioning Kit A25524
ProteomeLab IgY-Transferrin SC Proteome Partitioning Kit A25523
ProteomeLab IgY-HDL SC Proteome Partitioning Kit A25525
For information on our comprehensive line of ProteomeLab systems, please
contact your local Beckman Coulter representative or visit our web site at
www.beckmancoulter.com/proteomelab
* All trademarks are the property of their respective owners.
IgY affinity technology is licensed from Genway Biotech, Inc.
©2005 Beckman Coulter, Inc. Printed in U.S.A. on recycled paper.