ProteomicsWorkshop2014

Report
Targeted Proteomics
Kelly Stecker
Sussman 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 proteomics
MRM: Multiple Reaction Monitoring
SRM: Selective/Selected Reaction Monitoring
Specifically monitoring or ‘targeting’ one or more peptides
Shotgun/untargeted proteomics:
Coomassie stained gel
Targeted proteomics:
Western blot
1
2
3
MRM/SRM/Targeted proteomics
MRM: Multiple Reaction Monitoring
SRM: Selective/Selected Reaction Monitoring
Specifically monitoring or ‘targeting’ one or more peptides
Shotgun/untargeted proteomics:
Coomassie stained gel
-Indiscriminately
identifies most
abundant proteins
-No prior knowledge
required for protein
detection
-Information
obtained for a large
number of proteins
Targeted proteomics:
Western blot
1
2
3
-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.
Ion
Source
Detector
The DC and RF voltages are
tuned to stabilize particular m/z
ranges
www.waters.com
SRM analysis uses 2 stages of mass filtering
Q1
q2
Q3
Ion
Source
Detector
Fragmentation
Q1: Peptide mass is selected (parent ion)
q2: peptide is fragmented via collision induced dissociation
Q3: 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
q2
Q1
Q3
At1g01690.1
MSDALSAIPAAVHRNLSDKLYEKRKNAAL
MLENIVKNLTSSGDHDKISKVIEMLIKEFA
KSPQANHR NLTSSGDHDISK AQYLEQ
IVPPVINSFSDQDSRVRYYACEALY
SGDHDISK
Three transitions (aka 3 pieces of data identifying this peptide)
NLTSSGDHDISK
SGDHDISK
NLTSSGDHDISK
DHDISK
NLTSSGDHDISK
NLTSS
Basic workflow for SRM analysis
Extract proteins
Digest into peptides
Chromatographic separation of peptides (C18 column)
ESI
MS analysis
Q1
Parent
ion
selection
q2
Fragmentation
Q3
Fragment
ion
selection
Ion Intensity
Electrospray Ionization
Time
Peptides are quantified using stable
isotope labeled peptide standards
Endogenous: NLTSSGDHDISK
Standard: NLTSSGDHDIS[K+08]
Q1 mass
Q3 fragment
Q3 mass
637.67
y7
771.38
641.67
y7
779.38
Peptide A
Intensity
Peptide Standard
Endogenous
Endogenous
Single transition
m/z
Standard
Intensity
Extracted ion chromatogram (XIC)
Time
Peptide B
Peptides are quantified using stable
isotope labeled peptide standards
Endogenous: NLTSSGDHDISK
Standard: NLTSSGDHDIS[K+08]
m/z
Intensity
Extracted ion chromatogram (XIC)
Time
Overlay: Std & Endog.
Intensity
Endogenous
Q3 fragment
Q3 mass
637.67
y7
771.38
641.67
y7
779.38
Peptide A
Single transition
Peptide Standard
Q1 mass
Peptide B
Peptides can be multiplexed in a single
targeted MS run
Standard peptides
Endogenous peptides
Standards peptides
Endogenous
peptides
Peptide standards are spiked in during sample processing
Extract proteins
*
*
*
*
*
Digest into peptides
*
*
Chromatographic separation of peptides (C18 column)
* **
*
*
*
*
*
*
ESI
Single transition
MS analysis
Parent
ion
selection
q2
Fragmentation
Q3
Fragment
ion
selection
Intensity
Q1
Peptide Std.
Endogenous
m/z
Intensity
Electrospray Ionization
Time
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 Lab
https://skyline.gs.washington.edu/
Vendor specific software
also exists:
MultiQuant from ABSciex
How to select peptides for SRM analysis
Considerations
1. Feasibility of chemical synthesis
-Peptide length
(≤ 20 A.A., or ≤ 24 A.A.)
-PTMs?
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
Poor performing
Number of peptides
2. Physiochemical properties
-Hydrophobicity
-Chemically modified residues
(Met, Cys)
Good performing
Hydrophobicity bins (SSR Calc)
Picotti et. al. 2013 Nature Vol 494, pp 266-270
Examples of endogenous peptide detection
success rate
Sussman Lab data:
-Lab mate working with rat blood
proteins:
• In silico selection ~20%
• Empirical data ~80%
Success rate for peptide detection depending on selection source
-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.
Huttenhain et al. Sci Transl Med 11 July 2012: Vol. 4, Issue 142, p. 142ra94
Commercial options for peptide synthesis
Sigma-Aldrich
• PEPscreen Peptide libraries
• AQUA peptides
Thermo Scientific
• PEPotec
Pros
Cons
Guaranteed 7-day turn around
Cheap (around $60/peptide)
Length restriction (~20 amino acids)
Minimum order requirement (24)
More PTMs available
No minimum order size
>95% pure
Expensive ($200-$300/peptide)
Slow production (months)
Pros
Cons
Cheap! (around $40/peptide)
Minimum order requirement (4)
Peptides arrive resuspended
http://www.sigmaaldrich.com/life-science/custom-oligos/custom-peptides/product-highlights/pepscreen-peptides.html
http://www.piercenet.com/info/pepotec-srm-custom-peptide-libraries
Note: all prices are for heavy labeled peptides and are approximate
Developing SRM methods
What 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
MacCoss Lab
https://skyline.gs.washington.edu/
http://prospector.ucsf.edu/prospector/mshome.htm
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
Untargeted discovery data
Detection overlap between
samples
1400
Identificaitons
1200
Both
Exps
Single
Exp
Detection overlap between
injection replicates
Inj1
Offline SCX fraction +
4 hour LC-MS runs
Inj2
1000
800
306
600
460
185
No SCX fraction,
90min LC-MS runs
400
200
0
Protein
Peptide
PICC pS124
Intensity,
cps
PEN3 pS40
Intensity,
cps
Untargeted Quantitation
Unfractionate
SCX fraction
d
PEN3 pS40
Intensity
PICC pS124
Intensity
Comparison between MS methods
Time
Time
Targeted SRM Quantitation
Standard
Endogenous
peptide
peptide
Log2
(treated/control)
3 fold
Cold
FC
JA
Flg22
H2O2
ABA
KCl
NaCl
Mann.
+
1.2 fold +/3 fold -
AT5G56980 pS61
MSL9 pS124
EIF4A1 pT145
JAZ12 pS97
AHA1 pT948
AHA2 pT947
CPK5 pS552*
ERD14 pS59
YAK1 pY284
AHA3 pT882
AHA3 pT948
CAX4 pS38
PIP3B pS274
AHA4 pT959
PIP2F pS283
AMT1 pS488
AT5G53420 pS204
NIA1 pS537
NPC4 pT158
DaySleeper pS155
TRP1 pS214
PP2C-g pS347
RPS6 pS240
WDL1 pS6
ZAC pS155
AREB3 pS43
ABF2 pS86*
HSFB2B pS222
SnRK2.2 pS177
SnRK2.3 pS176
SnRK2.6.1 pS175
CPK9 pS78
PEN3 pS40
ADH1 pS229
CPK9 pT37
PEPC1 pS11
AHA2 pS899
Remorin pT58
SIP1 pS11
PICC pS124
Ox-reductase pS29
FAC1 pS203
PIP2F pS286
PLC2 pS280
GC5 pS793
V-ATPase pS241
SAY1 pS313
COP related pS24
MAP4Kα1 pS478
VCS pS692
bZIP30 pS176
MyoB1 pS825
PB1domain pS218
RAF18 pS671
TUA3 pT349*
TUA4 pT349*
SnRK2.4 pS158*
Vac14 pS624
Reliable peptide detection means
proteins can be reproducibly analyzed
across many different samples
ABA
responsive
block
Osmoticspecific
block
Heat map of 5 min phosphorylation
response of 60 peptides under 9
treatment conditions
Stecker et al. Plant Physiology 165.3 (2014): 1171-1187.
Log2
(treated/control)
3 fold
Cold
FC
JA
Flg22
H2O2
ABA
KCl
NaCl
Mann.
+
1.2 fold +/3 fold -
AT5G56980 pS61
MSL9 pS124
EIF4A1 pT145
JAZ12 pS97
AHA1 pT948
AHA2 pT947
CPK5 pS552*
ERD14 pS59
YAK1 pY284
AHA3 pT882
AHA3 pT948
CAX4 pS38
PIP3B pS274
AHA4 pT959
PIP2F pS283
AMT1 pS488
AT5G53420 pS204
NIA1 pS537
NPC4 pT158
DaySleeper pS155
TRP1 pS214
PP2C-g pS347
RPS6 pS240
WDL1 pS6
ZAC pS155
AREB3 pS43
ABF2 pS86*
HSFB2B pS222
SnRK2.2 pS177
SnRK2.3 pS176
SnRK2.6.1 pS175
CPK9 pS78
PEN3 pS40
ADH1 pS229
CPK9 pT37
PEPC1 pS11
AHA2 pS899
Remorin pT58
SIP1 pS11
PICC pS124
Ox-reductase pS29
FAC1 pS203
PIP2F pS286
PLC2 pS280
GC5 pS793
V-ATPase pS241
SAY1 pS313
COP related pS24
MAP4Kα1 pS478
VCS pS692
bZIP30 pS176
MyoB1 pS825
PB1domain pS218
RAF18 pS671
TUA3 pT349*
TUA4 pT349*
SnRK2.4 pS158*
Vac14 pS624
Reliable peptide detection means
proteins can be reproducibly analyzed
across many different samples
20%CV
Median
ABA
Cold
C1
8.5%
5.7%
12.9% 12.8% 6.8%
C2
C3
FC
Flg
H2O2
JA
KCl
Mann NaCl
5.8%
6.9%
6.5%
7.6%
10%
6.4%
9.3%
CV= Standard Deviation
Average
Cold
FC
JA
Flg22
H2O2
ABA
KCl
NaCl
Mann.
+
1.2 fold +/3 fold -
AT5G56980 pS61
MSL9 pS124
EIF4A1 pT145
JAZ12 pS97
AHA1 pT948
AHA2 pT947
CPK5 pS552*
ERD14 pS59
YAK1 pY284
AHA3 pT882
AHA3 pT948
CAX4 pS38
PIP3B pS274
AHA4 pT959
PIP2F pS283
AMT1 pS488
AT5G53420 pS204
NIA1 pS537
NPC4 pT158
DaySleeper pS155
TRP1 pS214
PP2C-g pS347
RPS6 pS240
WDL1 pS6
ZAC pS155
AREB3 pS43
ABF2 pS86*
HSFB2B pS222
SnRK2.2 pS177
SnRK2.3 pS176
SnRK2.6.1 pS175
CPK9 pS78
PEN3 pS40
ADH1 pS229
CPK9 pT37
PEPC1 pS11
AHA2 pS899
Remorin pT58
SIP1 pS11
PICC pS124
Ox-reductase pS29
FAC1 pS203
PIP2F pS286
PLC2 pS280
GC5 pS793
V-ATPase pS241
SAY1 pS313
COP related pS24
MAP4Kα1 pS478
VCS pS692
bZIP30 pS176
MyoB1 pS825
PB1domain pS218
RAF18 pS671
TUA3 pT349*
TUA4 pT349*
SnRK2.4 pS158*
Vac14 pS624
Reliable peptide detection means
proteins can be reproducibly analyzed
across many different samples
P-value
Log2
(treated/control)
3 fold
+/- 1.25 fold change
+/- 1.5 fold change
0.05
Students T-Test: 3 control
samples, 3 treated samples
Practical sample handling
comments
Three biological replicates
per treatment
Homogenization,
protein extraction
Spike in isotopically labeled peptide
standards, trypsin digest,
TiO2 phosphopeptide enrichment
It is difficult to correct for differential sample
handling before standard peptides are spiked in!
90 min LC-MS analysis using
Triple Quadrupole (QQQ)
Q1
Q2
Parent ion
selection
Q3
Fragmentatio
n
Fragment
ion selection
Intensity
Quantification of endogenous/standard
extracted ion chromatograms
Standard
Endogenou
s
m/z
Intensity
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
Targeted Proteomics
Time
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, pitfalls
and 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.

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