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volker.kruft@absciex.com
SWATH™: next generation data-independent (DIA) mass spectrometry for complete qualitative and
quantitative sample analysis
volker.kruft@absciex.com
Data-independent SWATH™ acquisition onAB SCIEX TripleTOF® mass spectrometers
volker.kruft@sciex.com
SWATH™: Recent advances in data-independent acquisition and the creation of the OneOMICS™
cloud ecosystem
volker.kruft@sciex.com
SWATH™: Recent advances in data-independent acquisition and the creation of the OneOMICS™
cloud ecosystem
5 © 2015 AB Sciex
Proteome 1 Proteome 2 Proteome 3 Proteome 4 Proteome 5 Proteome 6 Proteome 7
“omics”: it is all about time courses
6 © 2015 AB Sciex
“omics”: it is all about time courses
Proteome 1 Proteome 3 Proteome 4 Proteome 5 Proteome 6 Proteome 7Proteome 2
7 © 2015 AB Sciex
8 © 2015 AB Sciex
9 © 2015 AB Sciex
Mixed (“Chimeric”) MSMS Spectra
− Two co-eluting species identified from isotope patterns; separating monoisotopic peaks requires > 200K!
− MS/MS spectra would be mixed (“chimeric”) even if the precursors were detected in real-time
− Search engines don’t handle this well…
Precursor Window (Q1)
for MSMS
MNIENLK +2
FATHGGYLLQGK +3
Precursor Window (Q1)
for MSMS
2 peaks!
Note!Repeat analysis of the same E. coli
sample gave 10% ID variation!
10 © 2015 AB Sciex
Chimeric MSMS Spectrum
− Seems to affect 30-40% of all spectra – with 0.7 amu selection…
− …how much worse will it be at 3 amu or 4 amu− Really bad would be identifying a completely different
peptide/protein – never happens…− Does it?
MNIENLK +2 FATHGGYLLQGK +3
a1
Y9
Y10
Y11
Y7
Y8
11 © 2015 AB Sciex
12 © 2015 AB Sciex
SWATH™Conventional Proteomics
Targeted Proteomics
Conventional StrategiesDDA
SWATH™ StrategyDIA
Gold Standard QuantitationSRM/MRM
13 © 2015 AB Sciex
DDA vs. DIA vs. MRMDiscovery
(DDA)Targeted (MRM)
on almost everything
DIA
on targetson semi-random subset
Jarrett Egertson, MacCoss Lab
14 © 2015 AB Sciex
DDA vs. DIA vs. MRMDiscovery
(DDA)Targeted (MRM)
on almost everything
DIA
on targetson semi-random subset
Jarrett Egertson, MacCoss Lab
15 © 2015 AB Sciex
The goal: a complete picture
Jarrett Egertson, MacCoss Lab
16 © 2015 AB Sciex
DIA
semi-random subsets
Achieving the goal: data dependent acquisition?
Jarrett Egertson, MacCoss Lab
17 © 2015 AB Sciex
DIA
on targetson semi-random subset
Achieving the goal: MRM/SRM?
Jarrett Egertson, MacCoss Lab
18 © 2015 AB Sciex
DIA
on targetson semi-random subset
Achieving the goal: MRM/SRM?
Jarrett Egertson, MacCoss Lab
19 © 2015 AB Sciex
DIA
on targetson semi-random subset
Achieving the goal: data independent acquisition?
Jarrett Egertson, MacCoss Lab
on almost everything
20 © 2015 AB Sciex
Targeted (MRM)
Discovery (DDA)
on almost everything
DIA
on targetson semi-random subset
Achieving the goal: data independent acquisition?
Jarrett Egertson, MacCoss Lab
21 © 2015 AB Sciex
DIA
on targetson semi-random subset
Achieving the goal: MRM/SRM?
Jarrett Egertson, MacCoss Lab
22 © 2015 AB Sciex
DIA
on targetson semi-random subset
Achieving the goal: MRM/SRM?
Jarrett Egertson, MacCoss Lab
23 © 2015 AB Sciex
DIA
on targetson semi-random subset
Achieving the goal: MRM/SRM?
Jarrett Egertson, MacCoss Lab
Targeted (MRM)
24 © 2015 AB Sciex
1 amu 1 amu
1amu 0.01 amu 0.01 amu0.01 amu
MRM/SRM
MRMHR
Precursors FragmentsCID
MS/MS quantitation techniques
Triple quadrupole
TripleTOF™5600+
Transition
X amu
TripleTOF™5600+ SWATH-MS™
m/z m/z
How big can X be?
25 © 2015 AB Sciex
Q0 High Pressure Cell
LINAC®
collision cell
Accelerator TOF™ Analyzer
40 GHz Multichannel TDC Detector
Two-stage reflectron
30kHz Accelerator
15 kV Acceleration
voltage
Ion compression optics
QJet® Ion Guide
High Frequency
Q1
The AB SCIEX TripleTOF® Systems
26 © 2015 AB Sciex
Nature Methods - Method of the year 2012
27 © 2015 AB Sciex
February 6 (2013) AB Sciex press release
28 © 2015 AB Sciex
Sequential Window Acquisition of all THeoretical Fragment Ion Spectra (SWATH™)
Conceived at Ruedi Aebersold’s lab @ ETH ZürichImplemented on SCIEX TripleToF™ systemsData-Independent Acquisition (DIA)
29 © 2015 AB Sciex
Sequential Window Acquisition of all THeoretical Fragment Ion Spectra (SWATH™)
Conceived at Ruedi Aebersold’s lab @ ETH ZürichImplemented on SCIEX TripleToF™ systemsData-Independent Acquisition (DIA)
30 © 2015 AB Sciex
Sequential Window Acquisition of all THeoretical Fragment Ion Spectra (SWATH)
• Conceived at Ruedi Aebersold’s lab @ ETH Zürich• Implemented on AB SCIEX TripleToF 5600+ QqToF
• Data-Independent Acquisition (DIA) on the LC time scale with generic methods
• Focus on precursor ion swathes, instead of individual precursors
31 © 2015 AB Sciex
Sequential Window Acquisition of all THeoretical Fragment Ion Spectra (SWATH)
• Conceived at Ruedi Aebersold’s lab @ ETH Zürich• Implemented on AB SCIEX TripleToF 5600+ QqToF
• Data-Independent Acquisition (DIA) on the LC time scale with generic methods
• Focus on precursor ion swathes, instead of individual precursors
32 © 2015 AB Sciex
33 © 2015 AB Sciex
34 © 2015 AB Sciex
Sequential Window Acquisition of all THeoretical Fragment Ion Spectra (SWATH™)
Conceived at Ruedi Aebersold’s lab @ ETH ZürichImplemented on SCIEX TripleToF™ systemsData-Independent Acquisition (DIA)
35 © 2015 AB Sciex
400
500
600
700
800
900
1000
1100
1200
0
m/z
min10 20 30 40 50 60 70 80 90 100 110
SWATH-MS Acquisition Principle
Aebe
rsol
d an
d co
wor
kers
, ETH
Zur
ich
36 © 2015 AB Sciex
400
500
600
700
800
900
1000
1100
1200
0
m/z
min10 20 30 40 50 60 70 80 90 100 110
SWATH-MS Acquisition Principle
m/z range = [400-1200] � at 25Da per swath ó 32 swaths required� at 100ms per isolation window ó 3.2s cycle time
A swath = the ensemble of fragment ion spectra acquired through the chromatographic range and for a defined isolation window
Cycle time: time required to come back to the acquisition of the same isolation window
Aebe
rsol
d an
d co
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, ETH
Zur
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37 © 2015 AB Sciex
400
500
600
700
800
900
1000
1100
1200
0
m/z
min10 20 30 40 50 60 70 80 90 100 110
SWATH-MS Acquisition Principle
=> complete MS/MS maps for all the analytesin a single sample injection
Aebe
rsol
d an
d co
wor
kers
, ETH
Zur
ich
38 © 2015 AB Sciex
MRMHR Workflow
Time, min
**
*
39 © 2015 AB Sciex
MS/MSAll with SWATH Acquisition
Time, min
******
40 © 2015 AB Sciex
MS/MSAll with SWATH Acquisition
Time, min
41 © 2015 AB Sciex
SWATH™ Acquisition
MRM Workflow
High Res XICs
Targeted Quantitative WorkflowsTop Two Techniques
42 © 2015 AB Sciex
Gillet et al., MCP 2012, Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis.
Figure by Christina Ludwig
SWATH-MS: Data-independent acquisition with targeted data extraction
SRM TARGETED data
acquisition
precursor selection
fragmentation fragment selection
0.7 Da
Q1 Q3
0.7 Da
Q2
inte
nsity
time
SWATH-MS data-independent
acquisition
precursor selection
fragmentation scanning
25 Da
inte
nsity
time
Q1 TOF
time time
TARGETED data extraction
Q2
10 ppm
43 © 2015 AB Sciex
m/z
Ion
trans
mis
sion
Desired instrument performanceNormal transmission Transmission through 5600
m/z
Ion
trans
mis
sion
Independent control over the RF and DC power supplies on a resolving Quadrupole with novel methods for determining the “offset” provide accurate windows of isolation
44 © 2015 AB Sciex
m/z
Ion
trans
mis
sion
Normal transmission “SWATH” Transmission
m/z
Independent control over the RF and DC power supplies on a resolving quadrupole provides accurate windows of isolation
SWATH Hardware: Q1 isolation windows
Ion
trans
mis
sion
45 © 2015 AB Sciex
Transmission window of TripleTOF 5600
MS/MS Experiment of SWATH 874-900 without CE
TOF MS Experiment
Relative intraspectral intensities of TOF MS and SWATH MSMS experiments are similar, even at the edges of the SWATH mass window.
46 © 2015 AB Sciex46
TIC
MS1 map
SWATH-MS principle: Acquisition & Targeted analysis
Aebe
rsol
d an
d co
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, ETH
Zur
ich
47 © 2015 AB Sciex
Aebe
rsol
d an
d co
wor
kers
, ETH
Zur
ich
SWATH-MS principle: Acquisition & Targeted analysis
47
TIC
MS1 map
MS2/swath map [swath 600-625 m/z]
855.5395742.4556 671.4180 600.3368
327.1295
Peptide of interest
<<
<
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XICs of fragment ion traces:855.5395742.4556671.4180 600.3368 327.1295
<<
<
<<
<<
<
<
<
Aebe
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d an
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48 © 2015 AB Sciex
Targeted Analysis of SWATH-MS Data
Courtesy of Ruedi Aebersold
49 © 2015 AB Sciex
Comparison of Data Independent Acquisition methods
Publication Purvine et al.Proteomics 2003
Plumb et al.Rapid CommMass Spec 2006
Geiger et al. MCP 2010
Gillet et al. MCP 2012
Venable et al. Nat Meth 2004
Panchaud et al. Anal Chem 2011
First time a method combines the speed, sensitivity, resolution and specificity
Data Independent Acquisition (DIA)
Isolation width 25 Da 10 Da 2.5 Dano windows (800 Da)
MSE AIFShotgun CID SWATH DIA PAcIFICDIA method
Duty Cycle ~2s ~2s ~2s 3.2s ~4s 5.4s
Specificity 7% 7% 7% 89% 16% 68%
Dynamic range nd 3-4 logsacross LC
4 logsacross LC
4 logsintrascan
nd 7 logsacross LC
Nb injection(s)for full coverage
1 1 1 1 >4 >20
Instrumentation Q-TOF QqTOF Orbitrap QqTOF LTQ LTQ
Aebersold and coworkers, ETH Zurich
50 © 2015 AB Sciex
Specificity of SWATHSWATH-MS Specificity
Gillet et al. Mol Cell Prot, manuscript O111.016717
51 © 2015 AB Sciex
VTLTSEEEAR from Lactate Dehydrogenase
Peptide quantified over putative loading between 500 pg and 5 µg with R2 0.999, without replicates.
XICs from SWATH™ data.Data from Tom Knapman, AB SCIEX
52 © 2015 AB Sciex
First SWATH publications
53 © 2015 AB Sciex
Validation of predicted splice forms
Courtesy of Ruedi Aebersold
54 © 2015 AB Sciex
Validation of predicted splice forms
Courtesy of Ruedi Aebersold
55 © 2015 AB Sciex
A biological Question
56 © 2015 AB Sciex
Proof of principle: Extending the quantification analysis to 40 “more” mitochondrial proteins involved in the yeast respiratory chain (not initially anticipated)
SWATH-MS added value: quantification analysis extension
Condition 1: SWATH MS run 1
Condition 2: SWATH MS run 2 > 10 fold up 5-10 fold up 2-5 fold up 1-2 fold up 1-2 fold down > 2 fold down
ACP1
NDE2
NDI1
NDE1
Mitochondrial Matrix
Inter Membrane
Space
Cytosol “Complex I”
NADH dehydrogenase
Complex II succinate
dehydrogenase
SDH2 SDH1
SDH4 SDH3
Complex III ubiquinol
cytochrom-c reductase
Complex IV cytochrom-c
oxydase Complex V ATP synthase
PMA1
PMA2
IPP1
PPA2
PPi ATP + H2O
ATP1 ATP2 ATP3
ATP4 ATP5 ATP6
ATP7 ATP8 OLI1
ATP14 ATP15 ATP16
ATP17 ATP18 ATP19
ATP20 TIM11
RIP1 COB
BI2 BI3
CYB2
COR1 QCR2
QCR6 QCR7
QCR8 QCR9 QCR10
MCR1
COX1 COX2 COX3
AI4 AI5
COX4
COX5A COX5B
COX6 COX7 COX8 COX9
COX10
COX12 COX13 COX11
COX15 COX17 CCP1
HFA1 CTP1
ILV3 HEM15
DLD3
LYS4
LYS12 LYS21
ERG11 ILV5 ODC2
PRX1 MIR1
GRX5 NCP1
TRX3
Other mitochondrial proteins
GRX2
TCA cycle
NADH
Succinate
Not detected
COQ5
395 – 5’000 copies per cell
5’000 – 100’000 copies per cell
100’000 – 1E6 copies per cell
no abundance data
COQ9 COQ4
CYT1
CYB5
CBR1 CYC1 CYC7
Gillet et al. (2012) Mol Cell Prot manuscript O111.016717
57 © 2015 AB Sciex
Recent SWATH publications
58 © 2015 AB Sciex
BosentanPhenol
Hydroxy
Hydroxy-Phenol
MS/MSALL with SWATH™ AcquisitionTIC after Mass Defect Filter
Filter applied to all SWATH Acquisition windows (no additional filter based on mass region of interest)
59 © 2015 AB Sciex
Recent SWATH publications
60 © 2015 AB Sciex
46 SWATH posters and orals @ ASMS 2014
A novel SWATH-MS platform for comprehensive characterization of epigenetic histone modifications
AP-SWATH dynamic interactome of DJ-1 under oxidative stress: Implications for
Parkinson's Disease
Integration of SWATH and MRM for biomarker discovery of esophageal
squamous cell carcinoma
Combining derivatization and SWATH for the integrated quantification and identification
of aldehydes and ketones in biological samples
Differential Mobility Separation (DMS) to improve spectral correlation in
SWATHTM acquisition.Direct Non-Targeted Protein
Identification from SWATH Data Using Database Search
Discovery of Glycoprotein Signatures for Aggressive Prostate
Cancer via SWATH MS
Evaluation of SWATHTM as a diagnostic tool for Bacterial Identification Using a
Strain's Specific Library
Expansion of ion library for mining SWATH data through fractionation
proteomics
Harnessing the power of SWATH-MS for unbiased identification of O-
GlcNacylated proteins
61 © 2015 AB Sciex
SWATH publications – Nature Methods
62 © 2015 AB Sciex
SWATH publications – Mol Sys Biol
63 © 2015 AB Sciex
SWATH publications – Mol Sys Biol
64 © 2015 AB Sciex
Recent forensic publications
65 © 2015 AB Sciex
SWATH publications - HHE
66 © 2015 AB Sciex
Recent SWATH publications - HHE
PCA analysis
67 © 2015 AB Sciex
Recent SWATH publications - HHE
Protein/peptide candidates
68 © 2015 AB Sciex
Protein Expression Profiling Example
CDC37 HSP90A HSP90B
69 © 2015 AB Sciex
S L Y A S Sp P G G V Y A T Ry6 y5 y4 y3y8 y7
b3 b4 b5
S L Y A S S P G G V Y A T Ry6 y5 y4 y3y8 y7
b3 b4 b5
y7 y6 y5 y4 y3
b3 b4
Common transitionsy8 y7 y6 y5 y4 y3
b3 b4
Common transitionsy8
SWATH [700-725] SWATH [750-775]
Detection of phospho-peptide from the naked peptide transitionsSWATH-MS specials: peptide modification analysis – Phospho-peptides
70 © 2015 AB Sciex
S L Y A S Sp P G G V Y A T Ry6 y5 y4 y3y8 y7
b3 b4 b5
y12 y11 y10 y9
b7 b9
S L Y A S S P G G V Y A T Ry6 y5 y4 y3y8 y7
b3 b4 b5
y12 y11 y10 y9
b7 b9
Differential transitions
y12 y11 y10 y9 y8
y7 y6 y5 y4 y3
b3 b4
Common transitionsy8 y7 y6 y5 y4 y3
b3 b4
Common transitionsy8
b7 b9
Differential transitions
y12 y11 y10 y9 y8 b7 b9
SWATH [700-725] SWATH [750-775]
SWATH [700-725] SWATH [750-775]
Detection of phospho-peptide from the naked peptide transitionsSWATH-MS specials: peptide modification analysis – Phospho-peptides
Using SWATH™ Acquisition for Characterization and Quantification of the Epigenetic Histone Modifications
Sahana MollahHUPO 2014
72 © 2015 AB Sciex
ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHR…
SGRGKGGKGLGKGGAKRHRKVLR…
Jenuwein and Allis. Science, 2001, 293, 1074.
SGRGKQGGKARAKAK…
H3
H4
H2A
H2BPEPSKSAPAPKKGSKKAITKAQKKDGK…
ING HP1 Pc Eaf3
Histone acetylation (ac)-Transcriptional activation
Histone methylation (me1, me2 and me3)-Transcriptional activation or silencing
MethylationAcetylationphosphorylation
Luger et al. Nature, 389, 251-260, 1997.
Brd
Histone Code Hypothesis
• Specific set of histone PTMs function as a binding platform for recruiting proteins leading to transcriptional activation or inactivation
73 © 2015 AB Sciex
MS for Studying Histone ModificationsQualitative and Quantitative
GKGGKGLGKGGAK[Ac]R• MS/MS provides detailed
characterization of peptide sequence and PTM location
• Quantitation of various forms using unlabeled or labeled strategies
• Challenging for histone PTM study due to multiple isoforms
• Isobaric and difficult to separate by LC
Endogenous
Heavy labeled
74 © 2015 AB Sciex
Defining the Ideal Method for Histone PTM Analysis
• High Specificity required to resolve isoforms‒ MS/MS based strategy to obtain PTM location information‒ Narrow isolation window to ensure for better specificity for each isoform
• Highly Quantitative‒ Work on LC/MS time scale for when isoforms cannot be resolved‒ High reproducibility
• Broad Dynamic range‒ Good relative quantitation even when different isoforms have very different
occupancy levels
• Highly Multiplexed‒ Within a histone preparation, multiple protein forms and many different
modifications of interest
MS based Strategy
75 © 2015 AB Sciex
Data Independent Acquisition
ØMS/MSALL
• A data-independent workflow enabled by TripleTOF® system technology that use a Q1 isolation window to step across a mass range, collecting high resolution MS/MS spectra for all detectable analytes in a complex sample, in a single run
• Use variable Q1 isolation window with smaller window especially for m/z dense regions for better specificity
MS/MSALL with SWATH™ Acquisition
76 © 2015 AB Sciex
Smaller Q1 Window for better Specificity
• Reduce number of precursors in a window for increased specificity of quantification
• Set Q1 isolation window to 6 Da for MS/MS analysis
- A large number of modifiedhistones are 14 Da apart
(1Me, 2Me, 3Me, Ac). Use a Q1 window smaller than the + 2 charge state of the delta mass (7 Da)
6
The other aspect of increasing specificity is using site specific fragment ions from full scan MS/MS Data
77 © 2015 AB Sciex
Improving Specificity for Histone Isoforms
• Due to presence of different modification patterns with similar masses, tighter windows can in some cases provide improved specificity, reduced noise and provide better peak group detection
Narrow Q1 Window thru Variable Window SWATH™ Acquisition
25 Da x 100 msec 6 Da x 20 msec
Histone H4 (4-17): -GKGGKGLGKGGAK[Ac]R
More Selective Q1 window
GK[1Me]GGKGLGKGGAK[AC]R
78 © 2015 AB Sciex
K(3Me)STGGKAPR
K(Ac)STGGKAPR
KSTGGK(Ac)APR
Improving Specificity for Histone Isoforms
• Isoforms of H3 peptide (9-17) KSTGGKAPR
• MS level shows the presence of multiple peaks for m/z 528.314 +/0.03amu
• XIC of unique MS/MS fragment pattern is used to differentiate the isoforms
Using MS/MS Fragments for Specific PTM Localization
MS Data
MS/MS Data
KSTGGK(3Me)APR
MS PeakXIC of m/z 528.314 + 0.03
XIC of differentiatingMS/MS fragments
79 © 2015 AB Sciex
Distinguish Co-Eluting Histone Isoforms
• Selection of the key fragment ions is critical for differentiation
• MS/MS provides resolution of two isobaric forms that cannot be chromatographically resolved
SWATH™ Acquisition Fragment Ions can Differentiate
MS PeakXIC of m/z 768.9465 + 0.025
XIC of differentiatingMS/MS fragments
GKGGK[Ac]GLGKGGAKR
GKGGKGLGK[Ac]GGAKR
GKGGKGLGKGGAK[Ac]R
Modified GKGGKGLGKGGAKR
80 © 2015 AB Sciex
Quantitation of H4 peptideAcetylated Isoforms of H4 peptide: GKGGKGLGKGGKAR
Sample Peptide
MS/MS modified/unmodified
ratio (y9 ion)
MS/MS modified/unmodified
ratio (y7 ion)
%CV of sum of MS/MS fragments (triplicate
analysis)
Human H9 cell RA untreated GK[1AC]GGKGLGKGGAKR <0.01 <0.01 22.9GKGGK[1AC]GLGKGGAKR 0.10 0.08 13.4GKGGKGLGK[1AC]GGAKR 0.06 0.03 12.6GKGGKGLGKGGAK[1AC]R 0.38 0.32 9.6
4.3Human H9 cell RA treated GK[1AC]GGKGLGKGGAKR <0.01 <0.01 10.4
GKGGK[1AC]GLGKGGAKR 0.03 0.03 5.6GKGGKGLGK[1AC]GGAKR 0.01 0.01 6.7GKGGKGLGKGGAK[1AC]R 0.50 0.42 3.0
mouse TS cell undiff GK[1AC]GGKGLGKGGAKR <0.01 <0.01 6.6GKGGK[1AC]GLGKGGAKR 0.07 0.07 7.2GKGGKGLGK[1AC]GGAKR 0.11 0.09 4.8GKGGKGLGKGGAK[1AC]R 0.26 0.22 4.4
mouse TS cell diff GK[1AC]GGKGLGKGGAKR <0.01 <0.01 10.8GKGGK[1AC]GLGKGGAKR 0.06 0.05 3.6GKGGKGLGK[1AC]GGAKR 0.09 0.07 5.2GKGGKGLGKGGAK[1Ac]R 0.23 0.18 5.7
• Absolute quantitation by one point calibration by spiking in heavy form of peptide
81 © 2015 AB Sciex
Highly Multiplexed
Sample Modified PeptideMS/MS modified/unmodified ratio (y7
ion) Histone H3 (9-17) K[3Me]STGGKAPR 0.09
K[1Ac]STGGKAPR 0.05KSTGGK[1Ac]APR 0.21
H9 cell RA treated K[1Me]STGGK[Ac]APR 0.42K[2Me]STGGK[Ac]APR 0.01K[3Me]STGGK[1Ac]APR 0.03K[1Ac]STGGK[1Ac]APR 0.02K[1Me]STGGKAPR 1.53K[2Me]STGGKAPR 0.01KSTGGKAPR
K[3Me]STGGKAPR 0.05K[1Ac]STGGKAPR 0.10KSTGGK[1Ac]APR 0.45
H9 cell RA untreated K[1Me]STGGK[Ac]APR 0.49K[2Me]STGGK[Ac]APR 0.01K[3Me]STGGK[1Ac]APR 0.01K[1Ac]STGGK[1Ac]APR 0.06K[1Me]STGGKAPR 0.96K[2Me]STGGKAPR 0.01KSTGGKAPR
82 © 2015 AB Sciex
Conclusions
• SWATH™ provides MS/MS information for unambiguous assignment of modification sites
• Retention time MS profiles coupled with fragment ion XICs can disambiguate the different forms
- complete separation of histone isoforms is less critical
• A highly quantitative method with %CV similar to MRM quantitation
83 © 2015 AB Sciex
Acknowledgements
• University of Pennsylvania ‒ Benjamin Garcia‒ Zuo-Fei Yuan‒ Kelly R. Karch‒ Natarajan Bhanu ‒ Shu Lin
• AB SCIEX‒ Lei Xiong‒ Eric Johansen‒ Christie Hunter
84 © 2015 AB Sciex
SWATH™ Acquisition for MetabolomicsQualitative and Quantitative Analysis of Metabolites
− Linking LC-MS/MS data to Metabolomics Library
screening
XIC Manager can implement any AB
SCIEX library, list of chemical formulas or outputs from METLIN
software for metabolite screening
85 © 2015 AB Sciex
Acylcarnitine QuantitationXIC Manager for Targeted Extraction of MS or MS/MS
Acylcarnitine C18C25H49NO4
MS/MS for confirmation
86 © 2015 AB Sciex
Variations on a theme…
� Abundant protein has many non-specific cleavages� likely dependent on sample prep
and variable between samples
� What should be used for normalization?
� Which peptide should be used for quantitation?CIFAEMFRRKPLFCGNSEADQLGKIFDLIGLPPEDDWPRDVSLPRGAFPPRGPRPVQSVVPEMEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKDEGNPE
PPRGPRPVQSVVPEMEESGAQLLLEMLTFNPHK RGPRPVQSVVPEMEESGAQLLLEMLTFNPHK GPRPVQSVVPEMEESGAQLLLEMLTFNPHKR GPRPVQSVVPEMEESGAQLLLEMLTFNPHK GPRPVQSVVPEMEESGAQLLLEMLTFNPH PRPVQSVVPEMEESGAQLLLEMLTFNPHK RPVQSVVPEMEESGAQLLLEMLTFNPHK PVQSVVPEMEESGAQLLLEMLTFNPHKR PVQSVVPEMEESGAQLLLEMLTFNPHK PVQSVVPEMEESGAQLLLEMLTFNPH VQSVVPEMEESGAQLLLEMLTFNPHK SVVPEMEESGAQLLLEMLTFNPH SVVPEMEESGAQLLLEMLTFNPHK VVPEMEESGAQLLLEMLTFNPH VVPEMEESGAQLLLEMLTFNPHK PEMEESGAQLLLEMLTFNPHK EMEESGAQLLLEMLTFNPH EMEESGAQLLLEMLTFNPHK MEESGAQLLLEMLTFNPH MEESGAQLLLEMLTFNPHK EESGAQLLLEMLTFNPH EESGAQLLLEMLTFNPHK ESGAQLLLEMLTFNPH ESGAQLLLEMLTFNPHK SGAQLLLEMLTFNPHK SGAQLLLEMLTFNPH GAQLLLEMLTFNPHK GAQLLLEMLTFNPH AQLLLEMLTFNPHK LLLEMLTFNPHK LLLEMLTFNPH LLEMLTFNPH LEMLTFNPH SGAQLLLEML PVQSVVPEMEESGAQLLLEMLT GPRPVQSVVPEMEESGAQLLLEMLT PVQSVVPEMEESGAQLLLEML GPRPVQSVVPEMEESGAQLLLEML PVQSVVPEMEESGAQLLLEM GPRPVQSVVPEMEESGAQLLLEM PVQSVVPEMEESGAQLLLE GPRPVQSVVPEMEESGAQLLLE GPRPVQSVVPEMEESGAQLLL PVQSVVPEMEESGAQLLL GPRPVQSVVPEMEESGAQLL
IFAEMFR CIFAEMFRRKPLFCGNSEADQLGK AEMFRRKPLFCGNSEADQLGK RKPLFCGNSEADQLGK KPLFCGNSEADQLGK PLFCGNSEADQLGKIFDLIGLPPEDDWPR CGNSEADQLGKIFDLIGLPPEDDWPR GKIFDLIGLPPEDDWPR IFDLIGLPPEDDWPR IFDLIGLPPEDDWP IFDLIGLPPEDD IFDLIGLPPED IFDLIGLPP FDLIGLPPEDDWPR DLIGLPPEDDWPR LIGLPPEDDWPR IGLPPEDDWPR GLPPEDDWPR LPPEDDWPR PPEDDWPR
MATSRYEPVAEIGVGAYGTVYKARDPHSGHFVALKSVRVPNGGGGGGGLPISTVREVALLRRLEAFEHPNVVRLMDVCATSRTDREIKVTLVFEHVDQDLRTYLDK VPNGGGGGGGLPISTVR VPNGGGGGGGLPISTV PNGGGGGGGLPISTVR GGGGGGGLPISTVR
YEPVAEIGVGAYGTVYK DPHSGHFVALK YEPVAEIGVG YEPVAEIGVGAY EPVAEIGVGAYGTVYK PVAEIGVGAYGTVYK VAEIGVGAYGTVYK IGVGAYGTVYK
RLEAFEHPNVVR LEAFEHPNVVR EAFEHPNVVR AFEHPNVVR FEHPNVVR PNVVRLMDVCATSR TSRTDREIKVTLVFEHVDQDLR SRTDREIKVTLVFEHVDQDLR REIKVTLVFEHVDQDLR EIKVTLVFEHVDQDLR KVTLVFEHVDQDLR VTLVFEHVDQDLR VTLVFEHVDQD VTLVFEHVDQ VTLVFEHVD TLVFEHVDQDLR LVFEHVDQDLR VFEHVDQDLR FEHVDQDLR
APPPGLPAETIKDLMRQFLRGLDFLHANCIVHRDLKPENILVTSGGTVKLADFGLARIYSYQMALTPVVVTLWYRAPEVLLQSTYATPVDMWSVG APEVLLQSTYATPVDM APEVLLQSTYATPVD APEVLLQSTYATP APEVLLQSTYA APEVLLQSTY STYATPVDMWSVG
GLDFLHAN GLDFLHANCIVHR VHRDLKPENILVTSGGTVK RDLKPENILVTSGGTVK DLKPENILVTSGGTVK LKPENILVTSGGTVK KPENILVTSGGTVK PENILVTSGGTVK ENILVTSGGTVK ILVTSGGTVK
APPPGLPAETIK APPPGLPAETIKDLMR
IYSYQMAL IYSYQMALTPVVV IYSYQMALTPVVVT IYSYQMALTPVVVTLWYR YSYQMALTPVVVTLWYR SYQMALTPVVVTLWYR YQMALTPVVVTLWYR QMALTPVVVTLWYR MALTPVVVTLWYR MALTPVVVTLWYR ALTPVVVTLWYR LTPVVVTLWYR TPVVVTLWYR PVVVTLWYR VVVTLWYR ALTPVVVTLW ALTPVVVTL
LADFGLAR
� Select peptides for MRM analysis
� Monitor data for QC
87 © 2015 AB Sciex
SWATH Initiatives
88 © 2015 AB Sciex
Human Spectral LibraryLibraries: Public Domain
89 © 2015 AB Sciex
M. tuberculosis Spectral LibrarySRMAtlas
0
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0200400600800
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[%]
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in library
Libraries: Public Domain
90 © 2015 AB Sciex
M. tuberculosis Spectral LibrarySRMAtlas
0
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tein
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in library
Libraries: Public Domain
91 © 2015 AB Sciex
M. tuberculosis Spectral LibrarySRMAtlas
Libraries: Public Domain
http://www.srmatlas.org/mtb/swath.php
92 © 2015 AB Sciex
M. tuberculosis Spectral Library
� Covered additional proteins with synthetic peptides (MtbAtlas*)
� 38,482 unique proteotypic peptides
� 3,838 proteins ( 96% of the annotated proteome)
� Download library from SWATHAtlas website -http://www.srmatlas.org/mtb/swath.php
SRMAtlas
0
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f Pro
tein
s
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in library
Libraries: Public Domain
93 © 2015 AB Sciex
SRMAtlas
Libraries: from IDA experiments
94 © 2015 AB Sciex
Processing SWATH-MS/MS Data
Libraries: Theoretical
Appendix A: Text File Field Requirements and Descriptions The text file must be a tab-delimited .txt file containing all of the following fields. Field name
Description
Q1 Q1 m/z (precursor m/z) Q3 Q3 m/z (fragment m/z) RT_detected retention time isotype isotype type (for example, Heavy, Light, M00, M04,
M08). Default is Light. uniprot_id database accession number (regardless of the type
of database used, use “uniprot_id” as the field name)
relative_intensity fragment ion intensity stripped_sequence peptide sequences without modifications, for
example, DEYELLCLDGSR modification_sequence peptide sequences with modifications, for example,
DEYELLC[CAM]LDGS[Dhy]R prec_z peptide charge protein_name protein name frg_type fragment type (b or y ion) frg_z fragment charge frg_nr ion number (for example, y3 ion = 3)
95 © 2015 AB Sciex
Processing SWATH-MS/MS Data
Libraries: Theoretical
96 © 2015 AB Sciex
97 © 2015 AB Sciex
Processing SWATH-MS/MS Data� Same as MRMHR
– User tells software the mass of the parent ion, and the fragment ion– Software automatically extracts XIC from the appropriate SWATH scan
(i.e. the mass range containing the parent ion)
• MS/MS Library Purity Score • Mass Error• Isotopic Pattern• Retention Time
98 © 2015 AB Sciex
� Novel normalization scheme– compensates for differences; allows smaller changes to be detected
� Weighted statistics give fold change and confidence
Visualization
Proteins Peptides
Log fold change
2 sample comparison
Confidence
Each column compares 2 samples Each rectangle compares 2 samples
100 © 2015 AB Sciex
Variable Window SWATH™ Acquisition� Adjust Q1 selection window
maintain roughly constant number of peptides to maintain specificity
– Narrower window in m/z dense regions
– Optimal cycle time maintained by adjusting accumulation time
� Reduce number of precursors in window for increased specificity of quantification
� Data acquisition supported in Analyst® Software TF 1.7
101 © 2015 AB Sciex
Improving Specificity of Detection
� While many low level peptide detections are good, there are some that have interferences
� Tighter windows can in some cases provide improved specificity, reduced noise and provide better peak group detection
Variable Window SWATH™ Acquisition
Fixed windows - 24 Variable Windows - 40
16.0 16.5 17.0Time, min
0
300
16.30
15.5 16.0Time, min
0
300
Inte
nsity
, cps
GLNEEQGNVVSRMore Selective Q1 window
(25 Da)(11 Da)
102 © 2015 AB Sciex
Adjust Fragment Ions
� Automatic fragment ion selection is intensity driven
� Full scan data is present in SWATH™ Acquisition data, therefore different fragment ions can be chosen when required
� Using the Edit Transitions dialogue, select desired transitions
61.2 61.6 62.0 62.4Time, min
0
3000
600061.83
62.22
61.2 61.6 62.0 62.4Time, min
0
3000
600061.84
Inte
nsity
, cps
Inte
nsity
, cps
103 © 2015 AB Sciex
Digging deeper and Maintaining Quantitation Quality� More confident detections are found at
lower abundances with the more narrow Q1 windows– # of fragment ions from the 1% FDR
peptides plotted vs. their peak areas
� As depth of coverage increases, it is important to ensure this is not at the expense of quantitation quality
104 © 2015 AB Sciex
Leveraging Expanded Dynamic Range
� Key figures of merit:– 1% FDR cutoff = confident
detection– <20% CV quant quality cutoff
Higher Loads of yeast on TripleTOF® 6600 System
Increase Specificity
3 µg 120min VW_6022%3 µg 60min VW_60
1 µg 60min VW_60
31%
3 µg 120min VW_10020%
~22,100 peptides
~11,600 peptides
90% more peptide coverage Increase Signal
105 © 2015 AB Sciex
Highly Multiplexed Quantification with Variable Windows
SWATH™ Identification & Quantification Dashboard – 60 VW x 37msec
90% of peptides with <20% CV
2%30%
Low median CV’s across
intensity range
� E.coli lysate, 1 µg load, 60 min gradient, 75µm x 30cm cHiPLC® column
4 ordersdynamic range
106 © 2015 AB Sciex
107 © 2015 AB Sciex
Recent SWATH publications
108 © 2015 AB Sciex
SWATHTM vs label free MS1 quant in DDA‘’MRM like’ quality: improved specificity & sensitivity
MS1 traceClear interference
SWATH MS/MStrace
MS1 traceNot detected
SWATH MS/MStrace
Data courtesy of Ludovic Gillet ETH
109 © 2015 AB Sciex
Easy Building of Variable Window Methods
• Simple interface for automated acquisition method building1. Import text file for full control over acquisition windows2. Set MS and MS/MS parameters3. Automatically build variable window SWATH™ acquisition method
For Increased Specificity
Q1 Start Q1 Stop CES
1
2
3
Variable Window Calculator Tool
• Open TIC of target proteome and extract out a MS spectrum of the whole LC run
• Generate m/z vs intensity list to paste into Excel
• Set assay parameters and build method
• www.absciex.com\VariableWindowsCalculator‒ Includes E.coli histogram for getting started
Excel Sheet for Designing based on Constant Precursor Density
600 800 1000 1200 1400Mass/Charge, Da
0
15000
687.8690
577.2933
Inte
nsity
, cps
Averaged Averaged MS Spectrum
m/z vs. Intensity list
20 30 40Time, min
0
5e7
29.479
23.012
Inte
nsity
, cps
TOF MS TOF MS TIC
Computation Computation of Windows
111 © 2015 AB Sciex
Assessing Impact of Window Sizes
• Fixed window (400-1000m/z)‒ 24 windows - 25 Da x 90 msec‒ 30 windows - 20 Da x 70 msec‒ 40 windows - 15 Da x 54 msec
• Variable window (400-1250m/z)‒ 24 windows - VW x 90 msec‒ 30 windows - VW x 70 msec‒ 40 windows - VW x 54 msec‒ 60 windows - VW x 37 msec
• Replicate injections were performed with each method using E. colidigest
112 © 2015 AB Sciex
Improving Peptide Detection and Quantitation
• Filter peptides at 1% FDR then count peptides at 20% CV or better
• Decreasing window size (increased # of windows) provided more peptide detections at 1% FDR with high quality quantitation across replicates
• 35% gain at peptide level
• 21% gain at protein level
1% Peptide FDR
More Selective Q1 window
34% gain in peptides21% gain in proteins
113 © 2015 AB Sciex
Variable Window SWATH™ Acquisition
114 © 2015 AB Sciex
Variable Window SWATH™ Acquisition
115 © 2015 AB Sciex
Variable Window SWATH™ Acquisition
116 © 2015 AB Sciex
The Complexity of Systems Biology
metabolomicshormones
metabolismLesi
hman
ia
Amino acids
signaling
neurodegenerative disorders
cardiovascular
obesitymass spectrometryDMS
Receptor signaling
Mem
bran
e tr
affic
king
bioc
hem
istry
membrane protein receptor
lipidomics
epigenetics
inflammation
microRNAparasites
molecular biology
next-gen sequencingBa
cter
ial p
atho
gens
cancer
transcriptomics
DNAproteomics
tuberculosisProtein kinases
cytokines
Histones
Alzheimer’s disease
Prio
ns
SWATHOneOmics™
Leading to a systems-level understanding
117 © 2015 AB Sciex
OneOmics™ in the CloudNext-gen sequencing meets Next-gen proteomics
• Cloud computing‒ Simple and scalable‒ Universal access to data‒ Fast processing
• Next-gen proteomics‒ Quantify thousands of proteins‒ Excellent repeatability and quantitation‒ Data completeness >98%
• Next-gen sequencing‒ Fast and inexpensive‒ Highly accurate and repeatable‒ Comprehensive
118 © 2015 AB Sciex
The OneOmics™ Project
1
Omics integration
OMX @ SCXGenomics
TranscriptomicsNext-gen
sequencing (NGS)
Proteomics Next-gen proteomics (NGP)
SWATH® & immunoMRM
Lipidomics Next-gen lipidomics (NGL)
3
App Store innovation
2
Collaborate securely
Metabolomics
Next-gen metabolomics (NGM)
119 © 2015 AB Sciex
Illumina® BaseSpace®
• Web-based data management and analysis
• Eliminates need for onsite storage and computing power
• Tools for collaboration and sharing
• NEW Up to 50x faster than desktop processing of SWATH Proteomics
Omics? There are Apps for that.
120 © 2015 AB Sciex
AB SCIEX SWATH™ Proteomics Cloud Tool Kit
• Input: SWATH Acquisition data from TripleTOF® systems
• Processing: Extracts and integrates the peptide/protein peaks using an Ion Library
• Output: Generates peak area results files
Value – up to 30x faster processing
121 © 2015 AB Sciex
AB SCIEX SWATH™ Proteomics Cloud Tool Kit
• Input: Peak areas from Extractor
• Processing: Normalizes data and computes the differential protein expression levels between the samples. Annotated with important meta data.
• Output: Protein ratios between samples and confidence metrics.
Value – processing with study level considerations
122 © 2015 AB Sciex
AB SCIEX SWATH™ Proteomics Cloud Tool Kit
• Input: Peak areas from Extractor
• Processing: Normalizes data and computes the differential protein expression levels between the samples. Annotated with important meta data.
• Output: Protein ratios between samples and confidence metrics.
123 © 2015 AB Sciex
AB SCIEX SWATH™ Proteomics Cloud Tool Kit
• Input: Protein expression ratios and confidence metrics from Assembler
• Processing: Detailed review of protein expression data (protein/peptide level). Informative visualizations of protein differences between samples with biological annotation. Changes mapped at the protein and gene level, allowing modifications, splice variants, etc to be visualized.
• Output: Visualization plots, publication ready
Beta App
Value – high value figures reflecting biology
124 © 2015 AB Sciex
AB SCIEX SWATH™ Proteomics Cloud Tool Kit
• Input: Protein expression ratios and confidence metrics from Assembler
• Processing: Detailed review of protein expression data (protein/peptide level). Informative visualizations of protein differences between samples with biological annotation. Changes mapped at the protein and gene level, allowing modifications, splice variants, etc to be visualized.
• Output: Visualization plots, publication ready
Beta App
125 © 2015 AB Sciex
AB SCIEX SWATH™ Proteomics Cloud ToolkitProtein Expression Browser
126 © 2015 AB Sciex
AB SCIEX SWATH™ Proteomics Cloud Tool Kit
• Input: Protein expression ratios and confidence metrics from Assembler
• Processing: Detailed review of protein expression data. Informative visualizations of data reproducibility, data normalization and FDR analysis.
• Output: Visualization plots
Beta App
Value – results you can trust
127 © 2015 AB Sciex
From Genomics to Proteomics and Beyond
Gene Expression
SWATHIon library
ProteinPilotAB SCIEX
Protein Exp. AssemblerAB SCIEX
Protein Exp. ExtractorAB SCIEX
Protein Expression
Value – Serious Science!!
128 © 2015 AB Sciex
From Genomics to Proteomics and Beyond
Gene Expression
SWATHIon library
ProteinPilotAB SCIEX
Protein Exp. AssemblerAB SCIEX
Protein Exp. ExtractorAB SCIEX
Protein Expression
Combining Next Generation Proteomics and NGS through OneOmics™ to gain new insights into human spermatogenesis
charles.pineau@inserm.fr
130 © 2015 AB Sciex
Multi-Omics Analysis of Primary Cytotrophoblasts from Second Trimester and
Term Placentas
Katy Williams1, Christie L. Hunter2; Andrew Olson3
1University of California San Francisco, USA; 2SCIEX, USA; 3Advaita Biosciences, USA
For Research Use Only. Not for use in Diagnostic Procedures
131 © 2015 AB Sciex
For those wishing to keep their feet on the ground …
• Position desktop vs cloud? – 5 mins - ChristieSWATH™ Acquisition 2.0
Data Acquisition
Ion Library
XIC Generation
Profiling
Value – Industry Standard for DIA
132 © 2015 AB Sciex
For those wishing to keep their feet on the ground …
• Position desktop vs cloud? – 5 mins - ChristieSWATH™ Acquisition 2.0
Data Acquisition
Ion Library
XIC Generation
Profiling
133 © 2015 AB Sciex
SWATH Digital Recordrecord once - analyse for ever
134 © 2015 AB Sciex
Conclusions
• Pilot study for a combined workflow for Transcriptomics and Proteomics
• OneOmics™ in combination with iPathwayGuideprovided a seamless pipeline from data to biological answers‒ Fast data processing of SWATH® Acquisition -
Extractor‒ Study level quantitation with normalization and
fold change computation – Assembler‒ View protein expression changes and data
quality – Browser / Analytics‒ Determine biological significance –
iPathwayGuide
• BaseSpace cloud environment for secure collaboration
135 © 2015 AB Sciex
Conclusions
• designing OMICS experiments needs careful planning
• we can now describe samples in (nearly) complete details
• identification and quantitation can be done on one system
• speed @ sensitivity @ resolution will be the key
136 © 2015 AB Sciex
ThanksDanke
Merci
Hvala
Gracias
Grazie
Děkuji
Ačiu
Eucaristw
Dank
Tak
ありがとう
Tack
Obrigado
Спасибо
Tesekkurler
谢谢
감사 Kiitos
MulţumescKöszönöm
( شكریھ) بہت
Salamat po
תודה רבה
ขอบคุณБлагодаря!
Děkuij
дєкуюPaldies
Dziękuję
감사
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Гялайлаа ধন বাদ
Thank you for your attention
137 © 2015 AB Sciex
Thank you!
138 © 2015 AB Sciex
139 © 2015 AB Sciex
140 © 2015 AB Sciex
Questions ?
141 © 2015 AB Sciex
AB SCIEX MS/MSusers only
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Thank You
143 © 2015 AB Sciex
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