Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec...

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Targeted Proteomics Kelly Stecker Sussman Lab

Transcript of Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec...

Page 1: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Targeted Proteomics

Kelly SteckerSussman Lab

Page 2: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Outline

• What is MRM/SRM/Targeted mass spec• Quantification using peptide standards• Selecting standard peptides and building

methods• Practical notes and suggestions.

Page 3: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

MRM/SRM/Targeted proteomicsMRM: Multiple Reaction Monitoring SRM: Selective/Selected Reaction MonitoringSpecifically monitoring or ‘targeting’ one or more peptides

1 2 3

Shotgun/untargeted proteomics: Coomassie stained gel

Targeted proteomics: Western blot

Page 4: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

MRM/SRM/Targeted proteomicsMRM: Multiple Reaction Monitoring SRM: Selective/Selected Reaction MonitoringSpecifically monitoring or ‘targeting’ one or more peptides

1 2 3

Shotgun/untargeted proteomics: Coomassie stained gel

Targeted proteomics: Western blot

-Indiscriminately identifies most abundant proteins

-No prior knowledge required for protein detection

-Information obtained for a large number of proteins

-Selectively targets one protein

-Requires prior knowledge of protein mass/sequence

-Limited number of proteins can be assays at once (<150)

-Improved sensitivity!-Higher throughput!

Page 5: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Peptides are targeted using a triple quadrupole mass spectrometer (QQQ)

Triple quads contain 3 quadrupoles in series that are programed to selectively stabilize your ion of interest. Quadrupoles act as a mass filter.

www.waters.com

Ion Source

Detector

The DC and RF voltages are tuned to stabilize particular m/z ranges

Page 6: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

SRM analysis uses 2 stages of mass filtering

Ion Source

Detector

Fragmentation

Q1 q2 Q3

Q1: Peptide mass is selected (parent ion)q2: peptide is fragmented via collision induced dissociationQ3: Peptide fragment is selected (fragment ion)

• Parent ion to fragment ion mass change is called a “transition”• Usually ≥ 3 transitions are monitored for each peptide of interest

Page 7: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

SRM analysis uses 2 stages of mass filtering

Q1 q2 Q3

Three transitions (aka 3 pieces of data identifying this peptide)

MSDALSAIPAAVHRNLSDKLYEKRKNAALMLENIVKNLTSSGDHDKISKVIEMLIKEFAKSPQANHR AQYLEQIVPPVINSFSDQDSRVRYYACEALY

NLTSSGDHDISK

At1g01690.1 SK

DISK

DHDISK

SGDHDISK

NLTSS

NLTSSGDH

NLSGDHDISK

NLTSSGDHDISK SGDHDISK

NLTSSGDHDISK DHDISK

NLTSSGDHDISK NLTSS

Page 8: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Extract proteins Digest into peptides

Basic workflow for SRM analysisChromatographic separation of peptides (C18 column)

ESI

Electrospray Ionization

Parent ion selection

Fragment ion

selection

Fragmen-tation

Q1 q2 Q3

MS analysis

Ion

Inte

nsity

Time

Page 9: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Peptides are quantified using stable isotope labeled peptide standards

NLTSSGDHDISKEndogenous:

Standard: NLTSSGDHDIS[K+08]

Q1 mass637.67

641.67

Q3 fragmenty7

y7

Q3 mass

779.38

771.38

Single transition

Inte

nsity

Time

Inte

nsity

m/z

Endogenous

Peptide StandardE

nd

og

en

ou

s S

tan

da

rd

Peptide A Peptide B

Extracted ion chromatogram (XIC)

Page 10: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Peptides are quantified using stable isotope labeled peptide standards

NLTSSGDHDISKEndogenous:

Standard: NLTSSGDHDIS[K+08]

Q1 mass637.67

641.67

Q3 fragmenty7

y7

Q3 mass

779.38

771.38

Single transition

Inte

nsity

Time

Inte

nsity

m/z

Endogenous

Peptide Standard

Extracted ion chromatogram (XIC)

Peptide A Peptide B

Ove

rlay:

Std

& E

ndog

.

Page 11: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Peptides can be multiplexed in a single targeted MS run

Standards peptides

Endogenous peptides

Standard peptides

Endogenous peptides

Page 12: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Extract proteins Digest into peptides

Peptide standards are spiked in during sample processing

Chromatographic separation of peptides (C18 column)

ESI

Electrospray Ionization

Parent ion selection

Fragment ion

selection

Fragmen-tation

Q1 q2 Q3

MS analysis

*

** *

* *

***

***

**

*

*

Inte

nsity

Time

Inte

nsity

m/z

Endogenous

Peptide Std.

Single transition

Page 13: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Quantitation is achieved by measuring area under XIC curve

Endog Area

Std. Area

signal intensity normalized to peptide

standard

=

Page 14: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Awesome freeware exists for analyzing SRM data

MacCoss Labhttps://skyline.gs.washington.edu/

Vendor specific software also exists: MultiQuant from ABSciex

Page 15: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

How to select peptides for SRM analysisConsiderations1. Feasibility of chemical synthesis

-Peptide length (≤ 20 A.A., or ≤ 24 A.A.)-PTMs?

2. Physiochemical properties-Hydrophobicity-Chemically modified residues (Met, Cys)

3. Biological considerations-Is the peptide unique to 1 protein-Likelihood of trypsin misscleavage

4. PRESENCE OF EMERPICAL MS DATA!-Has your protein been detected

by MS?-Software for predicting

proteotypic behavior” of peptides is

“Not so good”-Dr. MacCoss

Picotti et. al. 2013 Nature Vol 494, pp 266-270

Good performing

Poor performing

Num

ber o

f pep

tides

Hydrophobicity bins (SSR Calc)

Page 16: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Examples of endogenous peptide detection success rate

Huttenhain et al. Sci Transl Med 11 July 2012: Vol. 4, Issue 142, p. 142ra94

Success rate for peptide detection depending on selection source

-Lab mate working with rat blood proteins: • In silico selection ~20%• Empirical data ~80%

-Targeting specific Arabidopsis protein:11 tryptic peptides selected from in silico prediction, 2 endogenous peptides detected after SCX fractionation AND extended LC gradient. • In silico selection ~18%

-Arabidopsis phosphopeptides:65 peptides selected from discovery shotgun proteomics data, 61 endogenous peptides detected.• Empirical data ~93%

NOTE: Isobaric tags may influence peptide behavior. Keep this in mind when viewing discovery data from iTRAQ or TMT experiments. In general, good quality MS1 spectra is a good indicator of SRM peptide performance.

Sussman Lab data:

Page 17: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Commercial options for peptide synthesis

Sigma-Aldrich• PEPscreen Peptide libraries

• AQUA peptides

ProsCons

Guaranteed 7-day turn around Length restriction (~20 amino acids)Cheap (around $60/peptide) Minimum order requirement (24)

More PTMs available Expensive ($200-$300/peptide)No minimum order size Slow production (months)>95% pure

Thermo Scientific• PEPotec

ProsCons

Cheap! (around $40/peptide)Minimum order requirement (4)Peptides arrive resuspended

Note: all prices are for heavy labeled peptides and are approximate

http://www.piercenet.com/info/pepotec-srm-custom-peptide-libraries

http://www.sigmaaldrich.com/life-science/custom-oligos/custom-peptides/product-highlights/pepscreen-peptides.html

Page 18: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Developing SRM methodsWhat you need to know

-Peptide parent mass and charge state-Fragment peptide masses and charge states-Highly recommend building SRM methods by first starting with peptide

standards

Resources

http://prospector.ucsf.edu/prospector/mshome.htm

MacCoss Labhttps://skyline.gs.washington.edu/

Page 19: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Developing SRM methods

Step 0: Successfully resolubilize lyophilized peptide standards. Recommend stepwise resuspension.

Step 1: Determine strongest transitions for each peptide (start with 5/peptide; method can be trimmed down to 3/peptide later on). If your instrument has an ion trap, this process is easier.

Step 2: Optimize collision energy (CE). This must be performed for every single transitions.

Step 3: Determine retention time of peptide. Using scheduled SRM methods significantly improves multiplexing capability.

Step 4: Look for endogenous peptides. Determine necessary pre-fractionation steps.

Page 20: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Why I like targeted MS: improved peptide detection

PIC

C p

S12

4P

EN

3 pS

40

Unfractionated

SCX fraction Standard peptide

Endogenous peptide

Targeted SRM Quantitation Untargeted Quantitation

Inte

nsity

Inte

nsity

Time Time

PIC

C p

S12

4P

EN

3 pS

40In

tens

ity, c

psIn

tens

ity, c

ps

185306 460

Inj1 Inj2

Detection overlap between samples

Protein Peptide

0

200

400

600

800

1000

1200

1400Both ExpsSingle Exp

Iden

tifica

itons

Detection overlap between injection replicates

Untargeted discovery data

Comparison between MS methods

Offline SCX fraction + 4 hour LC-MS runs

No SCX fraction, 90min LC-MS runs

Page 21: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Col

dF

CJA F

lg22

H2O

2

AB

AK

Cl

NaC

lM

ann.

AT5G56980 pS61MSL9 pS124EIF4A1 pT145JAZ12 pS97AHA1 pT948AHA2 pT947CPK5 pS552*ERD14 pS59YAK1 pY284AHA3 pT882AHA3 pT948CAX4 pS38PIP3B pS274AHA4 pT959PIP2F pS283AMT1 pS488AT5G53420 pS204NIA1 pS537NPC4 pT158DaySleeper pS155TRP1 pS214PP2C-g pS347RPS6 pS240WDL1 pS6ZAC pS155AREB3 pS43ABF2 pS86*HSFB2B pS222SnRK2.2 pS177SnRK2.3 pS176SnRK2.6.1 pS175CPK9 pS78PEN3 pS40ADH1 pS229CPK9 pT37PEPC1 pS11AHA2 pS899Remorin pT58SIP1 pS11PICC pS124Ox-reductase pS29FAC1 pS203PIP2F pS286PLC2 pS280GC5 pS793V-ATPase pS241SAY1 pS313COP related pS24MAP4Kα1 pS478VCS pS692bZIP30 pS176MyoB1 pS825PB1domain pS218RAF18 pS671TUA3 pT349*TUA4 pT349*SnRK2.4 pS158*Vac14 pS624

ABA responsive

block

Heat Map

Name

Colors

1.590.26-0.26-1.59

Heat Map

Name

Colors

1.590.26-0.26-1.59

3 fold +1.2 fold +/-3 fold -

Osmotic-specific

block

Log2 (treated/control)

Reliable peptide detection means proteins can be reproducibly analyzed

across many different samples

Heat map of 5 min phosphorylation response of 60 peptides under 9

treatment conditions

Stecker et al. Plant Physiology 165.3 (2014): 1171-1187.

Page 22: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Col

dF

CJA F

lg22

H2O

2

AB

AK

Cl

NaC

lM

ann.

AT5G56980 pS61MSL9 pS124EIF4A1 pT145JAZ12 pS97AHA1 pT948AHA2 pT947CPK5 pS552*ERD14 pS59YAK1 pY284AHA3 pT882AHA3 pT948CAX4 pS38PIP3B pS274AHA4 pT959PIP2F pS283AMT1 pS488AT5G53420 pS204NIA1 pS537NPC4 pT158DaySleeper pS155TRP1 pS214PP2C-g pS347RPS6 pS240WDL1 pS6ZAC pS155AREB3 pS43ABF2 pS86*HSFB2B pS222SnRK2.2 pS177SnRK2.3 pS176SnRK2.6.1 pS175CPK9 pS78PEN3 pS40ADH1 pS229CPK9 pT37PEPC1 pS11AHA2 pS899Remorin pT58SIP1 pS11PICC pS124Ox-reductase pS29FAC1 pS203PIP2F pS286PLC2 pS280GC5 pS793V-ATPase pS241SAY1 pS313COP related pS24MAP4Kα1 pS478VCS pS692bZIP30 pS176MyoB1 pS825PB1domain pS218RAF18 pS671TUA3 pT349*TUA4 pT349*SnRK2.4 pS158*Vac14 pS624

Heat Map

Name

Colors

1.590.26-0.26-1.59

Heat Map

Name

Colors

1.590.26-0.26-1.59

3 fold +1.2 fold +/-3 fold -

Log2 (treated/control)

Reliable peptide detection means proteins can be reproducibly analyzed

across many different samplesBox Plot

treatment

CV

50 %

45 %

40 %

35 %

30 %

25 %

20 %

15 %

10 %

5 %

0 %

ABA ColdControl_1Control_2Control_3 FC Flg22 H2O2 JA KCl Mannitol NaCl0.08495410.0569620.1289970.1277060.06835310.05805110.06943360.06535070.07601430.1002050.06465850.0930643Median

Box Plot

treatment

CV

Color bytreatment

ABAColdControl_1Control_2Control_3FCFlg22H2O2JAKClMannitolNaCl

Reference points:

Median20%CV

ABA Cold C1 C2 C3 FC Flg H2O2 JA KCl Mann NaCl8.5% 5.7% 12.9% 12.8% 6.8% 5.8% 6.9% 6.5% 7.6% 10% 6.4% 9.3%Median

Standard Deviation Average

CV=

Page 23: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Col

dF

CJA F

lg22

H2O

2

AB

AK

Cl

NaC

lM

ann.

AT5G56980 pS61MSL9 pS124EIF4A1 pT145JAZ12 pS97AHA1 pT948AHA2 pT947CPK5 pS552*ERD14 pS59YAK1 pY284AHA3 pT882AHA3 pT948CAX4 pS38PIP3B pS274AHA4 pT959PIP2F pS283AMT1 pS488AT5G53420 pS204NIA1 pS537NPC4 pT158DaySleeper pS155TRP1 pS214PP2C-g pS347RPS6 pS240WDL1 pS6ZAC pS155AREB3 pS43ABF2 pS86*HSFB2B pS222SnRK2.2 pS177SnRK2.3 pS176SnRK2.6.1 pS175CPK9 pS78PEN3 pS40ADH1 pS229CPK9 pT37PEPC1 pS11AHA2 pS899Remorin pT58SIP1 pS11PICC pS124Ox-reductase pS29FAC1 pS203PIP2F pS286PLC2 pS280GC5 pS793V-ATPase pS241SAY1 pS313COP related pS24MAP4Kα1 pS478VCS pS692bZIP30 pS176MyoB1 pS825PB1domain pS218RAF18 pS671TUA3 pT349*TUA4 pT349*SnRK2.4 pS158*Vac14 pS624

Heat Map

Name

Colors

1.590.26-0.26-1.59

Heat Map

Name

Colors

1.590.26-0.26-1.59

3 fold +1.2 fold +/-3 fold -

Log2 (treated/control)

Reliable peptide detection means proteins can be reproducibly analyzed

across many different samplesScatter Plot

Log2(AreaRatio)

Pva

lue

-3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

Scatter Plot

Log2(AreaRatio)

Pva

lue

Color byTreatment

ABAColdFCFlg22H2O2JAKClMannitolNaCl

+/- 1.25 fold change+/- 1.5 fold change

0.05

Students T-Test: 3 control samples, 3 treated samples

P-va

lue

Page 24: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Practical sample handling comments

Process all samples and controls in the SAME batch!-Extract proteins on the same day-Spike standards on the same day from the same aliquot

It is difficult to correct for differential sample handling before standard peptides are spiked in!

Homogenization, protein extraction

Spike in isotopically labeled peptide standards, trypsin digest,

TiO2 phosphopeptide enrichment

90 min LC-MS analysis using Triple Quadrupole (QQQ)

Time

Inte

nsit

y

m/zIn

tens

ity

StandardEndogenou

s

Parent ion selection

Fragment ion selection

Fragmentation

Q1 Q2 Q3

Targeted Proteomics

Three biological replicates per treatment

Quantification of endogenous/standard extracted ion chromatograms

Page 25: Targeted Proteomics Kelly Stecker Sussman Lab. Outline What is MRM/SRM/Targeted mass spec Quantification using peptide standards Selecting standard peptides.

Useful references• “A complete mass-spectrometric map of the yeast proteome applied to quantitative trait analysis.”Paola Picotti, (lots of authors) Reudi Aebersold (2013) Nature

• “Selected reaction monitoring–based proteomics: workflows, potential, pitfallsand future directions.” Paola Picotti & Ruedi Aebersold (2012) Nature Methods

• “Selected reaction monitoring for quantitative proteomics: a tutorial.” Vinzenz Lange, Paola Picotti, Bruno Domon and Ruedi Aebersold (2008) Molecular Systems Biology 4:222

Arabidopsis SRM data from our lab• Stecker KE et al. "Phosphoproteomic Analyses Reveal Early Signaling Events in the Osmotic Stress

Response." Plant Physiology 165.3 (2014): 1171-1187.

• Su SH et al. "Deletion of a tandem gene family in Arabidopsis: increased MEKK2 abundance triggers autoimmunity when the MEKK1-MKK1/2-MPK4 signaling cascade is disrupted." The Plant Cell Online 25.5 (2013): 1895-1910.