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1
Quantitative analysis of the
proteome
Stephen Barnes, PhD
BMG 744 Proteomics-Mass
Spectrometry
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Proteomics Data Standards
• 2005 MCP - Paris guidelines
• 2008 HUPO – MIAPE and mzML
• 2008 NCI - Amsterdam principles (6)
• 2011 NCI – Sydney
– For users of public data
– Reviewers of journals
– Multi-site projects with unpublished data
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Kissinger et al MCP 10:1-9, 2011
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Proteomics Data Standards
• Common descriptive terms • Sufficient experimental description • Data format • Data quality
– Mass accuracy (evidence of calibration) – Repeatability (technical and biological replicates) – False discovery rate (MRM and pseudoMRM) – Degeneracy of MRM – # of peptides to make a match
• Reference materials
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Kissinger et al MCP 10:1-9, 2011
Use of isotopes – ICAT (do/d8) and ICAT 13C0/
13C8
– d0/d10 propionic anhydride (N-terminal labeling)
– 15N/14N (whole cell labeling)
– 18O/16O (trypsin)
– iTRAQ labeling
• Non-isotope methods – Peptide coverage
– Classical triple quadrupole methods
Quantitative proteomics
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Isotope-coded affinity technology
This reagent reacts with cysteine-containing proteins (80-85% of proteome)
Labeling can be replacement of hydrogens (X) with deuterium, or better to
exchange 12C with 13C in the linker region (this avoids chromatography issues)
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Quantification from ESI-mass spectrum
Schmidt et al., Mol Cell Prot, 2003
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Time-dependent leucine
incorporation with SILAC
The cells are pre-labeled with leucine-d0. Leucine-d3 is added to the medium and cells sampled at various times later. The peaks annotated with d0 and d3 are the triply charged peaks of the peptide VAPEEHPVLLTEAPLNPK, which contains three leucines.
Ong et al.,
MCP 1:367,
2002
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Verifying absorption of phosphoproteins onto IMAC
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Elongation factor 2 OS=Homo sapiens GN=EEF2 PE=1 SV=4
DSVVAGFQWATK
H:L Ratio 0.8168
Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4
ILLAELEQLK
H:L Ratio 1.6427
Brossier et al. unpublished
18O-labeling
• Trypsin catalyzes the transfer of 18O in 18O-enriched water to both the carboxylate
oxygens of the C-terminus of tryptic
peptides
R-COOH R-C18O2H
• The peptides have an increase in mass of
4 Da
• Generally not considered a large enough
mass difference
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iTRAQ quantification
• The iTRAQ™ reagents
– React with Lys amino groups and each one adds 145 Da to the molecular weight of the peptide
– Fragmentation produces reporter ions from m/z 114, 115, 116 and 117
– Current iTRAQ kit contains 8 forms with reporter fragment ions of m/z 114, 115, 116, 117, 118, 119 and 121
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iTRAQ™ Reagent Design
Amine specific
PRG
Peptide Reactive
Group
Charged Neutral loss
Isobaric Tag (Total mass = 145)
Reporter Balance PRG
• Gives strong signature ion in
MS/MS
• Gives good b- and y-ion series
• Maintains charge state
• Maintains ionization efficiency
of peptide
• Signature ion masses lie in
quiet region
Reporter (Mass = 114 thru 117)
• Balance changes
in concert with
reporter mass to
maintain total
mass of 145
• Neutral loss in
MS/MS
Balance (Mass = 31 thru 28)
Isobaric Tag (Total mass = 145)
= MS/MS Fragmentation Site
Isobaric Tag
Total mass = 145
Reporter Group mass
114 –117 (Retains Charge)
Balance Group
Mass 31-28 (Neutral loss)
Amine specific peptide
reactive group (NHS)
N
N
O
O
N
O
O
Slide provided by
Applied Biosystems 18 1/25/12
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Other non-isotopic quantitative
methods in proteomics
The coverage (the number of peptides observed for a protein) is sensitive to the amount of the protein
– This can be used to calculate whether a treatment affects the abundance of a protein where fold-change > 2
– Applies to LC-MS (MUDPIT methods)
900 1520 2140 2760 3380 4000 Mass (m/z)
0
8401.3
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
2452.17 1366.82
1494.90
2646.33 1267.76 2164.05 2517.29
1388.78 2186.02 3141.45 1544.76 3163.46
2 peptides, 10 fmol
900 1520 2140 2760 3380 4000 Mass (m/z)
0
8401.3
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
2452.17 1366.82
1494.90
2646.33 1267.76 2164.05 2517.29
1388.78 2186.02 3141.45 1544.76 3163.46
7 peptides, 50 fmol
900 1520 2140 2760 3380 4000 Mass (m/z)
0
8401.3
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
2452.17 1366.82
1494.90
2646.33 1267.76 2164.05 2517.29
1388.78 2186.02 3141.45 1544.76 3163.46
10 peptides, 200 fmol
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Triple quad MRM analysis
Peptides of interest can be analyzed like small molecules – Choose the parent molecular ion, collide with argon gas
and select a unique fragment
Q1 Q2 Q3 Detector
- + + - -
- -
- - - - - -
Collision gas
N 2
Gas Sample solution
5 KV
• Multiple reaction ion scanning
First filter the [M-H]- molecular ion of the analyte (Q1)
Fragment the molecular ion with N2 gas (Q2)
Select a specific (and unique) fragment ion (Q3)
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Quantitation experiment for
biotinylated cytochrome c MRM analysis monitored in 50 channels
2 4 6 8 10 12 14 16 18 20 22 24 26 28
Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
4.5e5
11.47
Each colored peak represents a different biotinylated peptide
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Quantitative Accuracy: Myoglobin
Color Indicates Method Used
iTRAQ
ICPL
ICAT 18O Labeling
Label Free
Label Free + targeted SRM
2D-Gels (nonDIGE)
2D-DIGE
B/A
Rat
io
16
10
6
0
2D Gels Label Free Stable
Isotope
Labeling
Anticipated Mole
Ratio 10
14
12
8
4
2
A = 0.5 pmol
B = 5 pmol
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2008 ABRF Study - identification of three truncated peptides
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MALDI-TOF and nanoLC-tandem MS
Bioinformatics analysis
Signaling and protein complexes analysis
Quantitative MRM analysis
Gel-LC for protein separation
Workflow for generation of proteomics data
Microarray analysis Biological and
experimental knowledge
MudPIT 2D-DIGE and other
electrophoresis
microRNA analysis
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Multiple Reaction Monitoring
Q0 Q1 Q2 Q3
LIT
Q0 Q1 Q2 Q3
LIT
Fragment Peptide Y or B ion
• MRM methods are the gold standard for quantitative analysis of small molecules
− Currently performed on a triple quadrupole instrument − Each tryptic peptide ion is isolated in Q1, fragmented by collision in Q2 and a
specific fragment measured after filtration in Q3
• Proteotypic peptides can represent proteins (like oligonucleotides for DNA) − Generally a 8-aa peptide is unique − Multiple channels - 10-20 msec per channel
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HIF-1a in kidney cytosol by LC-MRM-MS
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Multiple reaction ion monitoring of Krebs cycle enzymes
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y10
y9 y8
y7
y6
626.3118
713.3464
y5
y4
363.2034
y3
261.1557
y2
227.1751 b2
b3
Summation of all MRM channels
Fragment intensities of individual ions derived from m/z 677.4
Full MSMS spectrum
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Verifying and quantifying C-truncation
• aA crystallin is supposedly processed to a 173aa form from the 196aa
translated product. Interestingly, what we see is the removal of an
interior 23aa peptide, so it must be differential splicing, not
posttranslational processing.
• Processed rat aA crystallin has a chymotrypsin cleavage site at 141Phe
• This peptide can be observed as a triply charged peptide
– FSGPKVQSGLDAGHSERAIPVSREEKPSSAPSS
• The C-truncations observed by mass spectrometry imaging are the
following:
– SGPKVQSGLD (truncation at 151)
– SGPKVQSGLDAGHSE (truncation at 156)
– SGPKVQSGLDAGHSER (truncation at 157)
– SGPKVQSGLDAGHSERAIPVSR (truncation at 163)
– SGPKVQSGLDAGHSERAIPVSREEKPS (truncation at 168)
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8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5
Time, min
2000
4000
6000
8000
10000
12000
14000
16000
18000
I n t e
n s i t y
DAY 21 Lens
DAY 50 Lens
DAY 100 Lens
8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5
Time, min
0.0e0
5.0e3
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
I n t e
n s i t y
Full Length αA-Crystallin
SGPKVQSGLDAGHSERAIPVSREEKPSSAPSS
10.2 10.4 10.6 10.8 11.0 11.2 11.4 11.6 11.8 12.0 12.2 12.4 12.6 12.8 13.0 13.2 13.4 13.6 13.8 Time, min
200
300
400
500
600
I n t e
n s i t y
XIC from I21 C.wiff (sample 1) - I21 C, Experiment 13, +TOF MS^2 of 524.8 (100 - 2000): 668.325 +/- 0.025 Da, Gaussian smoothed XIC from I50 C.wiff (sample 1) - I50 C, Experiment 13, +TOF MS^2 of 524.8 (100 - 2000): 668.325 +/- 0.025 Da, Gaussian smoothed XIC from I100 C.wiff (sample 1) - I100 C, Experiment 13, +TOF MS^2 of 524.8 (100 - 2000): 668.325 +/- 0.025 Da, Gaussian smoothed
α-Tubulin Sample Control Peptide
APVISAEKAY
11.3 11.4 11.5 11.6 11.7 11.8 11.9 12.0 12.1 12.2 12.3 12.4 12.5 12.6 12.7
Time, min
0
1000
2000
3000
4000
5000
6000
I n t e
n s i t y
XIC from I21 C.wiff (sample 1) - I21 C, Experiment 10, +TOF MS^2 of 461.8 (100 - 2000): 722.404 +/- 0.025 Da, Gaussian smoothed XIC from I50 C.wiff (sample 1) - I50 C, Experiment 10, +TOF MS^2 of 461.8 (100 - 2000): 722.404 +/- 0.025 Da, Gaussian smoothed XIC from I100 C.wiff (sample 1) - I100 C, Experiment 10, +TOF MS^2 of 461.8 (100 - 2000): 722.404 +/- 0.025 Da, Gaussian smoothed
Bovine Serum Albumin Loading Control Peptide: (1fmole/µl)
AEFVEVTK
SGPKVQSGLD
1-151 aa. Truncation
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Concatenation - making 13C-
labeled peptide internal standards
K
K
K
K
K
K
Convert peptide
sequences to
oligo DNA
sequences
Splice together the
individual oligo DNAs to
form a composite cDNA
Insert cDNA into a
plasmid and
recombinantly express
in bacteria in the
presence of Lys-13C615N2
NH2- COOH
K*
K* K*
K*
K*
K*
Treat with trypsin
Anderson & Hunter, 2005
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Quantitative peptide MRM-MS
• The albumin-depleted plasma proteome is mixed
with the composite 13C,15N-labeled protein
internal standard and then treated with trypsin
• The molecular ions (doubly charged) and the
specific y ions for each peptide and its labeled
form are entered into the MRM script one channel
at a time
• A single run may consist of 30 peptides in 60
channels
• Sensitivity is compromised by “sharing out”
measurement time, but can be compensated for
by carrying out nanoLC
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Advantage of a C-terminal
labeled lysine
186 301 448 505 642 755 886 987 1115 b ions
A D E F G H I M T K
1133 1062 948 833 686 629 492 379 248 147 y ions
186 301 448 505 642 755 886 987 1123 b ions
A D E F G H I M T K*
1141 1070 956 841 694 637 500 387 256 155 y ions
With the labeled lysine is at the C-terminus, only
the b10 ion contains the isotope atoms
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References for these talks (1)
• Annan RS, Hudleston MJ, Verma R, Deshaies RJ, Carr SA. A multidimensional electrospray MS-based approach to phosphopeptide mapping. Anal. Chem. 73:393, 2001.
• Flory MR, Griffin TJ, Martin D, Aebersold R. Advances in quantitative proteomics using stable isotope tags. Trends in Biotechnology 20: S23, 2002.
• Taupenot L, Harper KL, O’Connor DT. The chromogranin-secretogranin family. New Engl. J. Med. 348: 1134, 2003.
• Lam YW, Mobley JA, Evans JE, Carmody JF, Ho S-M. Mass profiling-directed isolation and identification of a stage-specific serologic protein biomarker of advanced prostate cancer. Proteomics 5: 2927, 2005.
• Lehmann WD, Krüger R, Salek M, Hung CW, Wolschin F, Weckwerth W. Neutral loss-based phosphopeptide recognition: a collection of caveats. J Proteome Res. 6:2866-73, 2007.
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Bibliography (2) • Ong SE, Mann M. Mass spectrometry-based proteomics turns
quantitative. Nature Chemical Biology. 1:252-262, 2005.
• Gruhler A, Schulze WX, Matthiesen R, Mann M, Jensen ON. Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry. Molecular & Cellular Proteomics. 4:1697-1709, 2005.
• Anderson L, Hunter CL. Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins. Molecular & Cellular Proteomics 5:573-588, 2006.
• Yao X, Freas A, Ramirez J, Demirev PA, Fenselau C. Proteolytic 18O labeling for comparative proteomics: model studies with two serotypes of adenovirus. Analytical Chemistry 73, 2836-42, 2001.
• Wang G, Wu WW, Zeng W, Chou C-L, Shen R-F. Label-Free Protein Quantification Using LC-Coupled Ion Trap or FT Mass Spectrometry: Reproducibility, Linearity, and Application with Complex Proteomes. Journal of Proteome Research 5: 1214-1223, 2006.
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Bibliography (3) • Kirkpatrick DS, CDenison C, Gygi SP. Weighing in on ubiquitin: the
expanding role of mass-spectrometry-based proteomics. Nature Cell Biology 7: 750-757 (2005).
• Knuesel M, Cheung HT, Hamady M, Barthel KKB, Liu X. A Method of Mapping Protein Sumoylation Sites by Mass Spectrometry Using a Modified Small Ubiquitin-like Modifier 1 (SUMO-1) and a Computational Program. Molecular and Cellular Proteomics 4:1626-1636 (2005).
• Nagaraj N, Wisniewski JR, Geiger T, Cox J, Kircher M, Kelso J, Pääbo S, Mann M. Deep proteome and transcriptome mapping of a human cancer cell line. Molecular Systems Biology 7: 548 (2011).
• Beck M, Schmidt A, Malmstroem J, Claassen M, Ori A, Szymborska A, Herzog F, Rinner O, Ellenberg J, Aebersold R. The quantitative proteome of a human cell line. Molecular Systems Biology 7: 549 (2011).
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