US HUPO 2006
Absolute Quantification of the Lower Abundance Proteome Through
Immunoaffinity Depletion of the Twenty Most Abundant
Proteins in Human Serum
Jeffrey J. Porter, Justin Wildsmith, Christopher D. Melm, Mark D. Schuchard,
Kevin M. Ray, Dian Er Chen, and Graham B.I. Scott
Abstract
Methods
Plasma proteomics holds great promise for the future of biomarker discovery, as
well as in vitro diagnostics. Although plasma is readily accessible for analysis, the
study of the plasma proteome is fundamentally limited by its vast dynamic range
(10 orders of magnitude). Low-abundance proteins, carrying great diagnostic
potential, are often obscured by the presence of high-abundance serum proteins.
In order to address this problem, Sigma-Aldrich has developed the ProteoPrep®
20, an antibody-based resin capable of depleting 97–98% of the total protein
from plasma. Depletion of these high-abundance proteins allows for visualization
of proteins co-eluting with, and masked by, the high-abundance proteins and
peptides, using LC-MS methods.
Absolute Quantification (Protein-AQUA™) is a targeted quantitative proteomics
technique that exhibits robust efficacy and is being increasingly utilized for a
wide variety of quantitative proteomics studies. By applying the ProteoPrep 20
for depletion of the most abundant proteins, we have enabled the identification
and absolute quantification of numerous lower- abundance proteins from human
serum. We believe this may potentially provide a multiplexed targeted approach
to the discovery of proteins holding biological or diagnostic significance.
Introduction
The study of the human plasma proteome is an area of great interest, especially
for the pharmaceutical potential of identifying disease biomarkers. Many proteins
of pharmaceutical interest appear at low concentrations in the plasma and are,
therefore, difficult to detect. Identification of potential biomarkers is especially
difficult due to the presence of higher- abundance proteins. Depletion of these
high-abundance proteins allows for visualization of proteins co-eluting with, and
masked by, the high-abundance proteins and peptides, using LC-MS methods. By
removing the high-abundance proteins, higher loads of lower-abundance plasma
proteins can then be separated on reverse phase columns, allowing detection
and quantification of low copy number proteins. The ProteoPrep 20 Plasma
Immunodepletion Kit has been developed for removal of 20 high-abundance
proteins from 8 mL of plasma. Depletion of these 20 high-abundance proteins
removes greater than 97% of the proteins in plasma and permits analysis of
20- to 50-fold more of each individual protein for improved visualization of
lower copy number proteins. By applying the ProteoPrep 20 for depletion of the
20 most abundant proteins, we have enabled the identification and absolute
quantification of numerous lower-abundance proteins from human serum.
Plasma Facts
Transferrin
IgG
Fibrinogen
Ceruloplasmin
IgA
α-2-Macroglobulin
Complement C4
Complement C3
IgM
Haptoglobin
High-Abundance Protein Depletion
Twenty high-abundance proteins were depleted from plasma using the
ProteoPrep 20 Immunodepletion Kit (PROT20). A large 3.7-mL prototype spin
column capable of depleting 100 mL of human plasma was used. The protein
concentration of whole and depleted plasma was determined using Bradford
assay (B6916), with BSA as the standard.
Tryptic Digestion
Samples of both whole and depleted plasma were prepared in 100 mg aliquots.
Each sample was diluted to 0.25 mg/mL in ammonium bicarbonate buffer.
Acetonitrile was added to a final concentration of
9% (v/v), and each sample was reduced and alkylated using the ProteoPrep
Reduction/Alkylation Kit (PROTRA). Proteomics Grade Trypsin (T6567) was
added at a concentration of 1% (w/w) and the sample incubated for 3 hours
at 37 °C. An additional aliquot of trypsin at 1% (w/w) was added and then
the sample further incubated at 37 °C overnight. The digests were dried to
completion in a SpeedVac™.
LC-MS/MS Analysis
An injection of a tryptically digested ProteoPrep 20 depleted plasma sample
(prepared as described in Methods) was made using an Agilent capillary 1100
HPLC. The reverse phase separation was performed on a
15 cm × 2.1 mm Discovery C18 column, using a 3-hour gradient. The mobile
phase consisted of formic acidified water and acetonitrile. The separation
system was coupled to a Thermo Finnigan LTQ linear ion trap mass spectrometer
set to perform tandem MS on the ten most abundant ions in each full scan
spectrum. After obtaining tandem MS results on an ion of interest, it was
added to an exclusion list to facilitate the interrogation of lower-abundance
ions. An LC-SRM method was developed corresponding to the protein targets
albumin (ideotypic peptide = FQNALLVR) and gelsolin (ideotypic peptide =
GASQAGAPQGR), as outlined in the Results section.
AQUA Analysis
An AQUA Peptide stock solution was prepared by dissolving isotopically labeled
versions of the two target peptides (FQNALLVR* and GASQAGAPQGR*,
respectively) in 0.1% TFA to a final concentration of 62.5 fmol/mL. Each digest
sample (whole and depleted plasma) was dissolved in 20 mL of the AQUA
Peptide stock solution. Samples were analyzed using an Agilent capillary 1100
HPLC coupled to a Thermo Finnigan LTQ linear ion trap mass spectrometer.
Using an LC-SRM method, the absolute quantity of each peptide (and
corresponding protein) was determined.
The Protein-AQUA Method
Complement
Factor B
Step 1:
AQUA Peptide Selection
Prealbumin
Complement
Factor H
α-1-Antitrypsin
Complement C9
VPQVSTPTLVEVSR
Select an optimal tryptic peptide and
stable isotope amino acid from the
sequence of your protein of interest
VPQVSTPTLVEVSR*
Order synthetic AQUA Peptide from
Sigma-Genosys
Complement C1q
Lipoprotein(a)
Complement C8
10%
1%
α-1-Acid
Glycoprotein
Apolipoprotein B
Apolipoprotein A1
Optimize LC-MS/MS separation
protocol for quantitation
Albumin
90%
91–99%
• The 10 most abundant proteins represent approximately 90% of the total protein mass in human plasma.
• The 22 most abundant proteins are said to represent approximately 99% of the total protein mass in
human plasma.
• The ProteoPrep 20 plasma immunodepletion column removes the 20 high-abundance plasma/serum
proteins listed below. These 20 proteins represent approximately 97% of the total human plasma
protein mass.
Albumin
IgGs
Transferrin
Fibrinogen
IgAs
a-2-Macroglobulin
IgMs
a-1-Antitrypsin
Complement C3
Haptoglobin
Apolipoprotein A1
Apolipoprotein A2
Apolipoprotein B
Acid-1-Glycoprotein
Ceruloplasmin
Equilibrate Column
Prepare Sample
Extract protein from biological
samples and add known quantity of
AQUA Peptide
Complement C4
Complement C1q
IgDs
Prealbumin
Plasminogen
Digest
Analyze by LC-MS/MS or MALDI to
quantitate protein of interest
Remove Storage Buffer
Centrifuge 5 sec
Centrifuge 30 sec
Step 2:
Implementation
Prepare Spin Column
4.0 mL Equilibration Buffer
Dilute 8 µL plasma to 100 µL
Sigma-Aldrich Biotechnology
P.O. Box 14508
St. Louis, MO 63178
*Labeled amino acid
Add Sample to Column
100 µL
Figure 1: Overview of the Protein-AQUA Method. This method was developed by Dr. Steve Gygi and
colleagues at Harvard Medical School [Stemmann O., Zou H., Gerber S. A., Gygi S. P., Kirschner M. W.;
Dual inhibition of sister chromatid separation at metaphase, Cell 2001, Dec 14, 107: 715-726]. Limited
use of this method is permitted under a licensing arrangement with Harvard Medical School.
Incubate 20 min
Centrifuge 60 sec
Concentrate Depleted
Samples
Wash Column
2 × 100 µL Equilibration Buffer
10 samples up to 300 µL
Centrifuge 60 sec
Depleted Plasma Sample
300 µL
Neutralize
Elute Bound Proteins
2.0 mL Elution Buffer
RT: 34.92
AA: 7337949
100
Results
33.67
90
80
80
67.46
78.37
83.17
80.91
12
11
54.37
68.57
10
100.38
Relative Abundance
9
A
A
97.01
106.11
95.41
8
Albumin Peptide in Depleted Plasma
70
Relative Abundance
Base Peak Chromatogram for Un-depleted plasma
52.23
Relative Abundance
70
AQUA peptide for Albumin FQNALLVR
RT: 34.76
AA: 268981
100
Albumin Peptide in Plasma
90
60
Endogenous
50
40
60
C
Endogenous
50
40
30
30
20
20
7
6
5
40.47
4
72.45
41.05
3
27.12
30.56
33.29
35.46
38.90
2
66.59
48.06
48.85
42.82
43.90
64.68
62.15
113.11
107.82
105.24
104.49
61.35
57.96
23.71 26.55
1
87.02
76.55
69.92
56.43
45.26
36.99
74.01
93.96
87.90
10
127.54
121.83
110.73
102.81
89.48
10
113.86
0
122.92
116.44
0
130.73 131.77
121.25
RT: 34.88
AA: 30983
0
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
130
135
100
Time (min)
RT: 34.73
AA: 27012
100
481.09
100
90
90
80
80
90
80
Relative Abundance
70
B
70
60
70
50
60
40
30
604.14
493.11
960.54
535.60 575.41
10
313.28
391.16 422.75
605.17 642.64
400
500
600
685.47
844.48
774.22
700
800
873.95
953.57
900
60
AQUA
50
D
AQUA
50
984.55
986.58
0
300
B
Full MS Scan
20
1041.65
1000
1128.50 1149.42
1100
1206.54
1200
1263.78
1322.19
1300
1393.96
1426.34
1497.29 1541.56
1400
1619.01 1644.60 1709.21
1500
1600
1700
1759.05
1836.38 1881.27 1906.24
1800
1900
1982.89
y+1
6
685.5
F: ITMS + c ESI d Full ms2 [email protected] [ 120.00-985.00]
40
40
30
30
20
20
2000
m/z
100
10
90
80
10
0
70
0
0
5
10
15
20
25
Time (min)
60
b +1
2
276.1
30
y +1
2
274.2
20
y +1
1
175.1
10
0
50
100
150
200
b +1
3
390.2
y +2
6
343.1
0
250
300
350
400
Tandem MS
y+1
4
500.4
y+1
3
387.3
y+2
7
407.3
y+1
5
571.4
b +1
5
574.3
b +1
4
461.2
450
500
550
600
b +1
6
687.4
650
700
b +1
7
786.3
750
800
850
Figure 2: Development of a Protein-AQUA LC-SRM for Absolute Protein Quantification. An
injection of a tryptically digested ProteoPrep 20 depleted plasma sample (prepared as described in
Methods) was made using an Agilent capillary 1100 HPLC coupled to a Thermo Finnigan linear ion trap
mass spectrometer. The base peak chromatogram (Figure 2A) shows a complex mixture of species that
may or may not have been chromatographically resolved. By harnessing the power of tandem MS, more
than 100 proteins were positively identified, with confidence, from the depleted plasma sample. Two of
these proteins (albumin and gelsolin) were chosen for further study. The full scan MS shown in Figure
2B illustrates the high abundance of the 481 Da doubly charged ion from one of the albumin peptides
identified. This sequence is then confirmed in Figure 2C with an abundance of y- and b-ions. This
experimental approach lays the groundwork for choosing an appropriate AQUA peptide in addition to
providing the necessary information for designing the SRM experiment. As shown in Figures 2B and 2C,
the doubly charged parent ion of this specific albumin peptide was selected for fragmentation and the two
most abundant y-ions present in the tandem MS were selected for detection.
XC Coverage
Score
(%)
100
Protein Identification
30 Neuronal kinesin heavy chain
10
29.58
90
47
31 WIP-related protein
10
24
3 Alpha-1-antichymotrypsin
precursor
4 Inter-alpha-trypsin inhibitor heavy
chain H1
40
24
32 ATP-binding cassette sub-family F
10
15
40
15
33 NAD(P)H dehydrogenase
[quinone] 1
10
6
5 Afamin precursor
40
24
34 Inhibin beta A chain precursor
10
33
6 Kininogen-1 precursor
40
23
35 Corticosteroid-binding globulin
precursor
10
2
7 Complement factor H precursor
30
16
36 Neuropeptide FF receptor 1
10
8
100
35
37 Tripartite motif protein 25
8
9
15
38 CD180 antigen precursor
8
5
50
31.01
40
RT: 26.78
AA: 270
30
25.69
30.87
31.14
31.96
33.17
23
39 CCAAT displacement protein
8
13
11 Gelsolin precursor
20
15
40 Cytoplasmic linker protein 170
8
15
12 Hemopexin precursor
20
30
8
25
13 Vitronectin precursor
20
27
41 Eukaryotic translation initiation
factor 2
42 Sodium channel protein type I
alpha subunit
8
5
14 Alpha-2-antiplasmin precursor
20
24
43 Signal peptide peptidase-like 2B
8
9
15 Inter-alpha-trypsin inhibitor heavy
chain H4
16 Angiotensinogen precursor
20
32
44 Acid ceramidase precursor
8
28
10
12
45 Glutamate receptor, ionotropic
kainate 3
8
16
17 AMBP protein precursor
10
9
46 Gephyrin
8
14
18 Alpha-2-HS-glycoprotein precursor
10
25
47 ARD1 subunit homolog
8
33
19 F-box only protein 31
10
17
48 Low-density lipoprotein receptor
protein 2
8
6
20 Prothrombin precursor
10
14
49 Troponin I, fast skeletal muscle
8
15
21 Plasma protease C1 inhibitor
precursor
22 Tubulin tyrosine ligase-like
protein 4
10
8
50 Integrin alpha-E precursor
8
10
10
7
51 TSC22 domain family protein 1
6
25
23 Heparin cofactor II precursor
10
11
52 Secretogranin-3 precursor
6
12
24 Beta-2-glycoprotein I precursor
10
17
53 BRCA1-associated protein
6
12
25 Isovaleryl-CoA dehydrogenase
10
8
54 Reelin precursor
4
4
26 BLOC-1 subunit
10
14
55 Threonine synthase-like 1
4
19
27 Pantothenate kinase 3
10
4
56 Ciliary dynein heavy chain 5
2
12
28 Contactin 3 precursor
10
8
57 Sorting nexin-19
2
9
29 Complement C5 precursor
10
9
Table 1: Proteins Identified in LC-MS/MS Analysis of ProteoPrep 20 Depletion Sample (not
identified in whole plasma analysis). The proteins listed above were confidently identified in the
ProteoPrep 20 depleted sample and were not detected in the whole plasma LC-MS/MS analysis. Using the
ProteoPrep 20 depletion technology, we have enabled the identification and absolute quantification of
these proteins. Data sets were searched using the SEQUEST algorithm in Bioworks 3.2. Filtration of protein
hits consisted of XCORR values >1.9, 2.2, and 3.75 for singly, doubly, and triply charged ions, as well as
delta CN > 0.1 and Rsp > 4.
30
35
40
45
RT: 26.68
AA: 4753
Gelsolin Peptide in
Depleted Plasma
Endogenous
60
C
50
40
20
27.44
10
0
0
RT: 26.81
AA: 10369
100
RT: 26.82
AA: 133133
100
90
80
80
70
AQUA
B
AQUA
60
60
50
50
40
40
30
30
20
20
10
D
10
0
22
20
20
25
Time (min)
30
28.51
23.38
0
10 Antithrombin-III precursor
15
70
90
20
10
29.18
60
10
30
5
80
Endogenous
70
70
8 Serum amyloid P-component
precursor
9 Histidine-rich glycoprotein
precursor
0
90
16
2 Apolipoprotein A-IV precursor
45
Gelsolin Peptide in Plasma
26.30
80
XC Coverage
Score
(%)
120
40
90
20
21
1 Inter-alpha-trypsin inhibitor heavy
chain H2
35
Figure 3: Protein-AQUA Analysis of Albumin in Whole Plasma and ProteoPrep 20 Depleted
Samples. Selected reaction monitoring (SRM) specifically targets MS detection of the selected parent that
produces specific daughter ions. Figures 3A (whole plasma) and 3C (depleted) depict the endogenous
albumin peptide, FQNALLVR, as the doubly charged ions fragment to produce two abundant y-ions. These
same y-ions are monitored in the AQUA peptide standard shown in Figures 3B (whole plasma) and 3D
(depleted); however the absolute mass-to-charge of both parent and daughter ions in the AQUA spike
are of higher mass due to the incorporation of a stable isotope amino acid. Quantitation is performed by
integrating the co-eluting peak areas shown here at 34.8 minutes. Protein-AQUA analysis of the samples
indicates 99.8% depletion of albumin using the ProteoPrep 20 Plasma Immunodepletion Kit, which is in
excellent concordance with previously obtained ELISA data.
A
Protein Identification
30
Relative Abundance
50
40
Relative Abundance
Relative Abundance
C
24
26
28
Time (min)
30
32
34
22
24
26
28
Time (min)
30
32
34
Figure 4: Protein-AQUA Analysis of Gelsolin in Whole Plasma and ProteoPrep 20 Depleted
Samples. SRM Analysis of the endogenous gelsolin peptide, GASQAGAPQGR, results in a complex
chromatogram in whole plasma (Figure 4A) with poor resolution and low signal-to-noise. When analyzed
in depleted plasma (Figure 4C), the endogenous gelsolin peptide is easily separated from interfering
species and its concentration readily calculated by comparison to the AQUA peptide internal standard
(Figures 4B and 4D).
Conclusions
Plasma proteomics holds great promise for the future of biomarker discovery,
as well as in vitro diagnostics. Although plasma is readily accessible for
analysis, the study of the plasma proteome is fundamentally limited by its vast
dynamic range (10 orders of magnitude). Low-abundance proteins, carrying
great diagnostic potential, are often obscured by the presence of highabundance serum proteins. By applying the ProteoPrep 20 for depletion of
the most abundant proteins, we have enabled the identification and absolute
quantification of numerous lower-abundance proteins from human serum. We
believe this combination of technologies may potentially provide a multiplexed
targeted approach to the discovery of proteins holding biological or diagnostic
significance.
References
1. Anderson, N. L.; Anderson, N. G. The Human Plasma Proteome. Mol. Cell.
Proteomics 2002, 1, 845.
2. Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Proc. Natl.
Acad. Sci. USA 2003, 100, 6940.
00751-021104