June 9th, 2013
Matthew J. Rardin
MS1 and MS2 crosstalk in label free quantitation of mass spectrometry data
independent acquisitions
MS2/SWATH
MS1 678.34++ m/z
568.98++ m/z
528.18+++ m/z
Cytosol
OM
IMS
Matrix
IM
PNAS 2013 110 (16), 6601-6606.
SIRT3 regulated mitochondrial lysine acetylation
• Type 2 Diabetes• Metabolic syndromes• High/low-fat diet• Aging• Cancer
• Mitochondria were purified from 5WT and 5KO mice by differential centrifugation and normalized to total protein
• Perform a trypsin digest with two replicates per mitochondrial sample to control for process variability
• Spike-in acetyl standard LVSSVSDLP(acK)R as a sample process loading control
• Two polyclonal antibody combination for enrichment of lysine acetylated peptides
• Two injection replicates per sample were ran on the Triple TOF 5600
• Label free “relative” quantitation using MS1 Filtering
• Published spectral library information in Panorama
Strategy for identification and quantitation of the SIRT3 regulatedacetylome in mouse liver mitochondria
PNAS 2013 110 (16), 6601-6606.
Mol Cell Proteomics 2012 11: 202-214.
• Follow a few peptide analytes to >3000 peptides
• Label-free• Skyline interface and tools• MS platform/manufacturer independent
(QqTOF, FT and ITs)*
Skyline MS1 FilteringA quantitative tool for discovery proteomics experiments
Samples (1, 2, 3,… N)
Mass Spectrometer
Peptide Search Engine(s)
Redundant Spectral Library
Peptide Ion Chromatograms (raw data)
Peak Integration
Relative Quantitation
PNAS 2013 110 (16), 6601-6606.
Peak Integration of the peptide ELQHHVKAcSVTAPYK+++
Label free quantitation using MS1 Filtering in Skyline
WT1 WT2
KO1 KO2
WT1 WT2
KO1 KO2
M
M+1
M+2
Peak Integration Peak Area Replicate View
Peak area replicates of ACSM1 K534
PNAS 2013 110 (16), 6601-6606.
Label free quantitation of KAc peptides in SIRT3-/-
• Quantitation of 2017 KAc sites
• Coefficient of variation of the peptide standard was 26% across 40 replicates
• Peptide area is normalized to spiked-in peptide standard
• Identified 266 sites on 136 proteins with greater than 2-fold increase in SIRT3-/- (p-value<0.01)
• Majority of sites were unchanged in the KO
• Quantitation of peptides from mitochondrial lysates showed no significant increase in protein expression
PNAS 2013 110 (16), 6601-6606.
OTC – Urea cycle
• >2x & p-value < 0.01
• <2x or p-value > 0.01
A
B
p-value < 0.05*, 0.01**
Targeted quantitation of acetyl peptides by Selected Reaction Monitoring (SRM) mass spectrometry
• Data collected on a 5500 QTRAP
• Spiked in 25 fmol of a heavy labeled synthetic peptide corresponding to peptides of interest
• SRM demonstrates similar results to MS1 Filtering
PNAS 2013 110 (16), 6601-6606.
Data Independent Acquisition (DIA) on a Triple TOF 5600
Adding MS1 quantitation to a DIAMS2 (SWATH) acquisition
1. What is the relationship between MS1 and MS2 quantitation in a DIA acquisition? -MS1 scan is acquired between each SWATH cycle
2. Is there value in combining MS1 analysis to SWATH MS2 data?
3. Are there cases when one method (MS1 or MS2) provides more accurate data than otherWhen is one data set compromised?
SWATH - YAPVAKAcDLASR
MS1 - YAPVAKAcDLASR
Simple Matrix - 25fmol b-galA
Slope 1.03
Slope 1.0
Complex Matrix – 300 ng digest B
Slope 0.87
Slope 0.51
SWATH - MVQKAcSLAR
MS1 - MVQKAcSLAR
Std. Concentration Curve 4 amol – 25 fmol
MS1 Filtering vs SWATH in Simple and Complex Matrix
*
Inferences increase in the MS1 Signal with sample complexity
*
Simple Matrix – 25 fmol -b galComplex Matrix – 300 ng protein digest* - Interference
• 6 technical replicates were acquired using either 100ng or 300ng of a protein digest
• The frequency of the peptide ratios in MS2 was tighter than MS1
• The average ratio in the MS2 was closer to the actual ratio; 3.11 vs 2.83 in the MS1 Filtering
• Sum of fragment ions in MS2 dramatically improves the distribution of ratios and p-values
• SWATH performed better then MS1 Filtering, but not as dramatically as we expected
Quantitative comparison of 250 peptides at a fixed ratio using MS1 and SWATH
A.
Exp
ecte
d
120
627_
000
2_m
ito_S
W_T
5_25
_3p2
5_re
p1
000
6...r
ep3
001
0...r
ep5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 605.9781+++precursor [M+1] - 606.3124+++precursor [M+2] - 606.6464+++
Exp
ecte
d
120
627_
000
2_m
ito_S
W_T
5_25
_3p2
5_re
p1
000
6...r
ep3
001
0...r
ep5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 605.9781+++precursor [M+1] - 606.3124+++precursor [M+2] - 606.6464+++
Libr
ary
120
627_
000
2_m
ito_S
W_T
5_25
_3p2
5_r
ep1
000
6...r
ep3
001
0...r
ep5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y9 - 1024.5132+y8 - 893.4727+y7 - 730.4094+y6 - 617.3253+y3 - 275.1714+
Libr
ary
120
627_
000
2_m
ito_S
W_T
5_25
_3p2
5_r
ep1
000
6...r
ep3
001
0...r
ep5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y9 - 1024.5132+y8 - 893.4727+y7 - 730.4094+y6 - 617.3253+y3 - 275.1714+
Lib
rary
1206
27_
000
2_m
ito_S
W_T
5_2
5_3p
25_
rep
1
0006
...re
p3
0010
...re
p5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y9 - 1024.5132+y8 - 893.4727+y7 - 730.4094+y6 - 617.3253+y3 - 275.1714+
Lib
rary
1206
27_
000
2_m
ito_S
W_T
5_2
5_3p
25_
rep
1
0006
...re
p3
0010
...re
p5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y9 - 1024.5132+y8 - 893.4727+y7 - 730.4094+y6 - 617.3253+y3 - 275.1714+
y6-interference
expected rep1
SWATH MS2
MS1
B. Stable Interference
Exp
ecte
d
120
627_
000
2_m
ito_S
W_T
5_25
_3p2
5_re
p1
000
6...r
ep3
001
0...r
ep5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 605.9781+++precursor [M+1] - 606.3124+++precursor [M+2] - 606.6464+++
Exp
ecte
d
120
627_
000
2_m
ito_S
W_T
5_25
_3p2
5_re
p1
000
6...r
ep3
001
0...r
ep5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 605.9781+++precursor [M+1] - 606.3124+++precursor [M+2] - 606.6464+++
expected rep1
Library 120110_0006_Mito100_300_2 0007...300_3 0009...3000_2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y13 - 1443.7227+y12 - 1372.6856+y11 - 1225.6171+y10 - 1111.5742+y9 - 996.5473+
Libr
ary
1201
10_0
006
_Mito
100_
300_
2
0007
...30
0_3
0009
...30
00_2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y13 - 1443.7227+ y12 - 1372.6856+y11 - 1225.6171+ y10 - 1111.5742+y9 - 996.5473+
SWATH MS2
C. Weak MS2 signal
Library 120110_0006_Mito100_300_2 0007...300_3 0009...3000_2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y13 - 1443.7227+y12 - 1372.6856+y11 - 1225.6171+y10 - 1111.5742+y9 - 996.5473+
Libr
ary
1201
10_0
006
_Mito
100_
300_
2
0007
...30
0_3
0009
...30
00_2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y13 - 1443.7227+ y12 - 1372.6856+y11 - 1225.6171+ y10 - 1111.5742+y9 - 996.5473+
Library 120110_0006_Mito100_300_2 0007...300_3 0009...3000_2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y13 - 1443.7227+y12 - 1372.6856+y11 - 1225.6171+y10 - 1111.5742+y9 - 996.5473+
Libr
ary
1201
10_0
006
_Mito
100_
300_
2
0007
...30
0_3
0009
...30
00_2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y13 - 1443.7227+ y12 - 1372.6856+y11 - 1225.6171+ y10 - 1111.5742+y9 - 996.5473+
Exp
ecte
d
120
110_
000
6_M
ito10
0_30
0_2
000
7...3
00_3
000
9...3
000_
2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 943.0125++precursor [M+1] - 943.5140++precursor [M+2] - 944.0154++
Exp
ecte
d
120
110_
000
6_M
ito10
0_30
0_2
000
7...3
00_3
000
9...3
000_
2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 943.0125++precursor [M+1] - 943.5140++precursor [M+2] - 944.0154++
MS1
Exp
ecte
d
120
110_
000
6_M
ito10
0_3
00_2
000
7...3
00_3
000
9...3
000_
2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 943.0125++precursor [M+1] - 943.5140++precursor [M+2] - 944.0154++
Exp
ecte
d
120
110_
000
6_M
ito10
0_3
00_2
000
7...3
00_3
000
9...3
000_
2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 943.0125++precursor [M+1] - 943.5140++precursor [M+2] - 944.0154++
Exp
ecte
d
120
110_
000
6_M
ito10
0_30
0_2
000
7...3
00_3
000
9...3
000_
2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 943.0125++precursor [M+1] - 943.5140++precursor [M+2] - 944.0154++
Exp
ecte
d
120
110_
000
6_M
ito10
0_30
0_2
000
7...3
00_3
000
9...3
000_
2
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
precursor - 943.0125++precursor [M+1] - 943.5140++precursor [M+2] - 944.0154++
expected rep1
expected rep1
y8-interference
Exp
ect
ed
120
627_
0002
_mit
o_S
W_T
5_2
5_3p
25_r
ep1
000
6...
rep3
001
0...
rep5
Replicate
0
20
40
60
80
100
Pe
ak A
rea
Pe
rce
nta
ge
precursor - 599.7648++precursor [M+1] - 600.2662++precursor [M+2] - 600.7675++
Exp
ect
ed
120
627_
0002
_mit
o_S
W_T
5_2
5_3p
25_r
ep1
000
6...
rep3
001
0...
rep5
Replicate
0
20
40
60
80
100
Pe
ak A
rea
Pe
rce
nta
ge
precursor - 599.7648++precursor [M+1] - 600.2662++precursor [M+2] - 600.7675++
Lib
rary
1206
27_0
002
_mito
_SW
_T5
_25
_3p2
5_r
ep1
0006
...re
p3
0010
...re
p5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y9 - 996.4633+ y8 - 833.3999+y7 - 734.3315+ y9 - 498.7353++b3 - 366.1296+
Lib
rary
1206
27_0
002
_mito
_SW
_T5
_25
_3p2
5_r
ep1
0006
...re
p3
0010
...re
p5
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
erc
en
tag
e
y9 - 996.4633+ y8 - 833.3999+y7 - 734.3315+ y9 - 498.7353++b3 - 366.1296+
MS1
Dynamic Interference
SWATH MS2
expected rep1 rep2 rep3
expected rep1 rep2 rep3
Inferences in the MS2 Signal
Reducing interferences in the MS2 scan during a SWATH DIA
SWATH 25 m/z
MS1 scan – 250 msec
MS2 scan – 125 msec
SWATH 10 m/z
MS1 scan – 250 msec
MS2 scan – 50 msec
SUMMARY• During a SWATH DIA the MS1 scan is still acquired on the Triple TOF
5600.
• In Skyline, both the MS1 and MS2 scans can be extracted and used for quantification of peptide analytes
• In a simple matrix the MS1 scan appears to have greater sensitivity then the MS2 scan. However in more complex samples the MS2 scan outperforms the MS1 scan.
• The MS1 scan provides useful information and can be used as a secondary quantitation strategy
• Use of both scans allow for screening and identifying various types of interferences
• Reduction of the SWATH window in a complex sample reduces interferences without loss of sensitivity in the MS2 scan
Acknowledgements
Birgit Schilling – MOA 3:50pmMS1 Filtering for quantifying lysine
acetylation in E. coli.
Alexandria D’Souza – TP521 External tool development in Skyline
Jason Held – WP555Quantifying cysteine oxidation using MS1
Filtering and SWATH
Anna Zawadzka – THOD 8:50amBreast cancer biomarker study using
MS1 Filtering
Laboratory of Bradford W. Gibson
University of WashingtonMichael J. MacCossBrendan MacLean
MM+1M+2
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