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BMG 744 ProteomicsBMG 744 Proteomics--Mass Mass SpectrometrySpectrometry

Quantitative analysis of the proteome

Stephen Barnes, PhD

sbarnes@uab.edu

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Proteomics Data Standards

• 2005 MCP ‐ Paris guidelines• 2008 HUPO – MIAPE (Minimum Information About a Proteomics Experiment) and mzML

• 2008 NCI ‐ Amsterdam principles– (1) timing, (2) comprehensiveness, (3) format, (4) deposition to repositories, (5) quality metrics, and (6) responsibility for proteomics data release.

• 2011 NCI – Sydney– For users of public data– Reviewers of journalsReviewers of journals– Multi‐site projects with unpublished data

• 2013 HUPO Proteomics Standards Initiative– http://www.psidev.info/

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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• 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 isotopesICAT (d /d ) and ICAT 13C /13C

Quantitative proteomics

– ICAT (do/d8) and ICAT 13C0/13C8

– d0/d10 propionic anhydride (N-terminal labeling)

– 15N/14N (whole cell labeling)– 18O/16O (trypsin)– iTRAQ labelingg

• Non-isotope methods– Peptide coverage– Classical triple quadrupole methods (MRM)

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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 ESIQuantification from ESI--mass spectrummass spectrumSchmidt et al., Mol Cell Prot, 2003

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Quantification with isotopicallylabeled amino acids

D3

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Arginine‐13C/15N isotopic labeling

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Isotope labeling with 13C15N‐lysine

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1/28/13 15Brossier et al. unpublished

Verifying absorption of phosphoproteins onto IMAC 

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Inte

nsi

ty

DSVVAGFQWATK

H:L Ratio  0.8168

Elongation factor 2 OS=Homo sapiens GN=EEF2 PE=1 SV=4

ty

ILLAELEQLK

H:L Ratio  1.6427

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Inte

nsit

Vimentin OS=Homo sapiens GN=VIM PE=1 SV=4

Brossier et al. unpublished

1818OO--labelinglabeling

• Trypsin catalyzes the transfer of 18O in 18O enriched water to both the carboxylate18O-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 D4 Da

• Generally not considered a large enough mass difference

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Tandem mass tag reagents

• TMT reagents are isobaric, i.e., they have the same molecular weight and are chemicallysame molecular weight and are chemically the same, but their “parts” have different masses

• Some reagents have four parts:– A mass reporter (different for each reagent)

A l bl i– A cleavable region

– An isotopic balancing region

– A lysine‐NH2 reacting reagent

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iTRAQ quantificationiTRAQ quantification• The iTRAQ™ reagents

React with Lys amino– 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 117and 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

Charged Neutral loss

Isobaric Tag(Total mass = 145)

Reporter Balance PRG

• Gives strong signature ion in • Balance changes pg gMS/MS

• Gives good b- and y-ion series• Maintains charge state • Maintains ionization efficiency

of peptide• Signature ion masses lie in

quiet region

Balance changes in concert with reporter mass to maintain total mass of 145

• Neutral loss in MS/MS

Isobaric Tag(Total mass = 145)

= MS/MS Fragmentation SiteIsobaric Tag

Total mass = 145

PRG

Peptide Reactive Group

Reporter(Mass = 114 thru 117)

Balance(Mass = 31 thru 28)

Reporter Group mass114 –117 (Retains Charge)

Balance GroupMass 31-28 (Neutral loss)

Amine specific peptidereactive group (NHS)

N

N

O

O

N

O

O

Slide provided by Applied BiosystemsSlide provided by Applied Biosystems211/28/13

TMT reagent from Pierce

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A 6‐plex TMT reagent

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MS/MS spectrum of TMT tags

Th f th t ti tid hThe mass of the tryptic peptide when reacted with anyone of the TMT reagents is the same.

However, each reagent gives a separate reporter mass (m/z 126, 127, 128, 129, 130 and 131.

Therefore samples from different

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Therefore, samples from different experimental conditions can be combined and analyzed in a single run.

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Other nonOther non--isotopic quantitative isotopic quantitative methods in proteomicsmethods in proteomics

The coverage (the g (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 th b d f

8401.3

30

40

50

60

70

80

90

100

% In

tens

ity

2452.171366.82

1494 90

2 peptides, 10 fmol

8401.3

30

40

50

60

70

80

90

100

% In

tens

ity

2452.171366.82

1494 90

7 peptides, 50 fmol

8401.3

30

40

50

60

70

80

90

100

% In

tens

ity

2452.171366.82

1494 90

10 peptides, 200 fmol

the abundance of a protein where fold-change > 2

– Applies to LC-MS (MUDPIT methods)

900 1520 2140 2760 3380 4000Mass (m/z)

00

10

20

30 1494.90

2646.331267.76 2164.052517.291388.78 2186.02 3141.451544.76

3163.46

900 1520 2140 2760 3380 4000Mass (m/z)

00

10

20

30 1494.90

2646.331267.76 2164.052517.291388.78 2186.02 3141.451544.76

3163.46

900 1520 2140 2760 3380 4000Mass (m/z)

00

10

20

30 1494.90

2646.331267.76 2164.052517.291388.78 2186.02 3141.451544.76

3163.46

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This quote comes from the January 2013 issue of Nature Methods. It noted there are several q ymethods for measuring proteins (antibodies, immunofluorescence, protein arrays)

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Triple quad MRM analysisTriple quad MRM analysisPeptides of interest can be analyzed like small molecules

Collision gas

N2

Gas

Q1 Q2 Q3 Detector

‐++‐‐

‐‐

‐‐‐ ‐‐ ‐

Collision gasGasSamplesolution

5 KV

• Multiple reaction ion scanning

First filter the [M+nH]n+ precursor ion of the analyte (Q1)

Fragment the precursor ion with N gas (Q2)

• Multiple reaction ion scanning

First filter the [M+nH]n+ precursor ion of the analyte (Q1)

Fragment the precursor ion with N gas (Q2)Fragment the precursor ion with N2 gas (Q2)

Select a specific (and unique) product ion (Q3)

Measure ion current reaching the detector for 20-50 msec

Repeat with a precursor/product ion combination for another peptide, etc.

Fragment the precursor ion with N2 gas (Q2)

Select a specific (and unique) product ion (Q3)

Measure ion current reaching the detector for 20-50 msec

Repeat with a precursor/product ion combination for another peptide, etc.

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Multiple reaction ion monitoring

Quantitative analysis of peptides in a complex mixture carried out using a triple 

LCquadrupole instrument

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Q1                              Q2                            Q3           DetectorIonizer

Based on precursor ion/product ion pair(s)Courtesy, John Cutts

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Flight path of ions through the quadrupoleion mass is higher than the set mass

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Flight path of ions through the quadrupoleion mass is lower than the set mass

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Flight path of ions through the quadrupoleion mass is the same as the set mass

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The real quadrupole ions0.7 m/z wide

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Quantitation experiment for Quantitation experiment for biotinylatedbiotinylated cytochrome c cytochrome c

MRM analysis monitored in 50 channelsMRM analysis monitored in 50 channels

4 5e5

1.0e5

2.0e5

3.0e5

4.0e5

4.5e5

11.47

2 4 6 8 10 12 14 16 18 20 22 24 26 28

Time, min

0.0

Each colored peak represents a different biotinylated peptide

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Quantitative Accuracy: MyoglobinQuantitative Accuracy: Myoglobin

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2D Gels Label FreeStable Isotope Labeling

14

A = 0.5 pmolB = 5 pmol

Color Indicates Method Used

iTRAQ

ICPL

ICATICAT

B/A Ratio

10

6

Anticipated Mole Ratio 10

14

12

8

18O Labeling

Label Free

Label Free + targeted SRM

2D-Gels (nonDIGE)

2D-DIGE

0

4

2

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MALDI‐TOF and nanoLC‐tandem MS

Gel‐LC for protein separation

Workflow for generation of proteomics data

MudPIT2D‐DIGE and other electrophoresis

Bioinformatics analysis

Signaling and protein complexes analysis

Microarray analysisBiological and 

experimental knowledge

microRNA analysis

Quantitative MRM analysis

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HIFHIF‐‐11 in kidney cytosol by LCin kidney cytosol by LC‐‐MRMMRM‐‐MSMS

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Multiple reaction ion monitoring of Krebs cycle enzymes

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Splicing generates a new sequence at the junction between exon 4 and exon 6

Exon 4

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Exon 6

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Lee FJ et al., 2011

Limitations of MRM‐MS

• A single precursor/product ion combination is not sufficiently specific (see class on MRMPath)sufficiently specific (see class on MRMPath)

– Need 3‐4 product ions to provide specificity

– This decreases the number of different peptides that can be monitored per second

• The quadrupole analyzer has a low mass accuracy

– Typically the peak is passed through a 0.7m/z filterTypically the peak is passed through a 0.7 m/z filter

• In an ideal world, we need an MS instrument that can collect high mass accuracy (2‐3 ppm) MSMS spectra in 20‐50 msec

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Pseudo MRM Analysis

Select PeptideDetect All Fragments

Fragment peptide

• High resolution TOF Analyzer for

Q1 Q2

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• High resolution TOF Analyzer for detection of fragment ions TOF

Pseudo MRM Analysis

Select PeptideDetect All Fragments

Fragment peptide

• High resolution TOF Analyzer for

Q1 Q2

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• High resolution TOF Analyzer for detection of fragment ions TOF

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Pseudo MRM Analysis

Select PeptideDetect All Fragments

Fragment peptide

• High resolution TOF Analyzer for

Q1 Q2

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• High resolution TOF Analyzer for detection of fragment ions TOF

Pseudo MRM Analysis

Select PeptideDetect All Fragments

Fragment peptide

• High resolution TOF Analyzer for

Q1 Q2

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• High resolution TOF Analyzer for detection of fragment ions TOF

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Pseudo MRM Analysis

Select PeptideDetect All Fragments

Fragment peptide

TOF MS/MS Spectrum 

Q1 Q2

p

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• The key difference between the TripleTOF and the triple quad is that the entire MSMS spectrum is collected by the TripleTOF in a single 50 sec (or shorter) data acquisition – the selection of product ion to follow is made post‐data acquisition

TOF

Summation of all MRM channels

Fragment intensities of individual ions derived from m/z677.4

y10

y9y8

y7

y6

626.3118

713.3464

y5

y4

363.2034

y3261.1557

y2

227.1751b2

b3Full MSMS spectrum

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Verifying and quantifying C‐truncation

• A 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 processingposttranslational processing.

• Processed rat A 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)

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– SGPKVQSGLDAGHSE (truncation at 156)

– SGPKVQSGLDAGHSER (truncation at 157)

– SGPKVQSGLDAGHSERAIPVSR (truncation at 163)

– SGPKVQSGLDAGHSERAIPVSREEKPS (truncation at 168)

Fragmentation of a chymotrypticpeptide

NH2-SGPKVQSGLD-COOH[M+2H]2+ = 494.55

b-ions y-ionsb1 = - y1 = (134)b2 = 145 y2 = (247)b3 = 242 y3 = (304)b4 = 370 y4 = (391)b5 = 469 y5 = (519)b6 = 597 y6 = (618)6 y6 ( )b7 = 684 y7 = 746b8 = 741 y8 = 843b9 = 854 y9 = 900b10 = 969 y10 = 987

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16000

18000

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

Inte

nsi ty

DAY 21 Lens

DAY 50 Lens

DAY 100 Lens

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 smoothedXIC 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 I21 C.wiff (sample 1) - I21 C, Experiment 10, +TOF MS^2 of 461.8 (100 - 2000): 722.404 +/- 0.025 Da, Gaussian smoothedXIC 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

SGPKVQSGLD

1‐151 aa. Truncation 

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0.0e0

5.0e3

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

Inte

nsi ty

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 13.8Time, min

200

300

400

500

600

Inte

nsity

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 smoothedXIC 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 12.7Time, min

0

1000

2000

3000

4000

5000

6000

Inte

nsi ty

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 smoothedXIC 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

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Concatenation Concatenation -- making making 1313CC--labeled peptide internal standardslabeled peptide internal standards

K

KC t tid

Splice together the individual oligo DNAs to

K

K

K

K

Convert peptide sequences to oligo DNA sequences

form a composite cDNA

Insert cDNA into a plasmid and

bi tlNH2- COOH

recombinantly express in bacteria in the presence of Lys-13C6

15N2

K*

K*K*

K*

K*

K*

Treat with trypsin

Anderson & Hunter, 2005521/28/13

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Quantitative peptide MRMQuantitative peptide MRM--MSMS

• The albumin-depleted plasma proteome is mixed with the composite 13C,15N-labeled protein i t l t d d d th t t d ith t iinternal 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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The future – SWATH MS

• http://www.youtube.com/watch?v=VZAZtA_qEbEbg

• Data independent analysis with a mass spectrometer that has a fast enough analyzer (TOF) to allow comprehensive quantitative(TOF) to allow comprehensive quantitative analysis of ALL peptides that elute from a LC column 

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References for these talks (1)• Flory MR, Griffin TJ, Martin D, Aebersold R. Advances in quantitative

proteomics using stable isotope tags. Trends in Biotechnology 20: S23, 2002.

• Ong SE Mann M Mass spectrometry-based proteomics turnsOng 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 g p pserotypes 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 Bibliography (2)(2)• 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).

• Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M. Global quantification of mammalian gene expression control. Nature 473: 337-342 (2011).

• Picotti P, Bodenmiller B, Aebersold R. Proteomics meets the scientific method. Nat Methods. 10:24-7 (2011).

• Gillette MA, Carr SA. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat Methods. 10:28-34 (2013).

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Methods. 10:28 34 (2013). • Fonslow BR, Stein BD, Webb KJ, Xu T, Choi J, Park SK, Yates JR

3rd. Digestion and depletion of abundant proteins improves proteomic coverage. Nat Methods. 10:54-6 (2013).