targeted proteomics kelly stecker sussman lab. outline what is mrm/srm/targeted mass spec...
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Targeted Proteomics
Kelly SteckerSussman Lab
Outline
• What is MRM/SRM/Targeted mass spec• Quantification using peptide standards• Selecting standard peptides and building
methods• Practical notes and suggestions.
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
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!
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
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
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
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
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)
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
.
Peptides can be multiplexed in a single targeted MS run
Standards peptides
Endogenous peptides
Standard peptides
Endogenous 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
Quantitation is achieved by measuring area under XIC curve
Endog Area
Std. Area
signal intensity normalized to peptide
standard
=
Awesome freeware exists for analyzing SRM data
MacCoss Labhttps://skyline.gs.washington.edu/
Vendor specific software also exists: MultiQuant from ABSciex
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)
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:
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
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/
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.
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
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.
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=
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
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
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.