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SWATH XIC & SRM Workflows
29.11.2013Hannes Röst & George Rosenberger
ETH Zürich, IMSB, Aebersold / Malmström
LC-MS/MS ProteomicsMethods
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LC-MS/MS Proteomics
Measurement in three dimensional space of RT-MS1-MS2• Shotgun: Continuous measurement of MS2 dimension
(spectrum)• SRM/MRM: Continuous measurement of RT dimension
(XIC)• SWATH: Continuous measurement of RT and MS2
dimension (sequential windows) Creation of n multiplexed MS2 “maps” and one MS1 map (here n=32)
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11/29/13
DIA with SWATH MS
● 1. SWATH MS Acquisition (AB Sciex 5600)
– 1 MS1, 32 MS2 (25 Da windows, 400-1200 Da)
– 3.3 s cycle time → 30 – 40 s peak width● 2. Targeted Analysis method
– Extract transitions from spectral library → XIC
AB Sciex: http://www.absciex.com/applications/biomarker-discovery-and-omics-research/msmsall-with-swath-acquisition
4Gillet et al. (2012) Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Molecular and Cellular Proteomics. 11 (6).
SWATH MS: Acquisition
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SWATH MS: MS2 Map
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SWATH MS: XIC
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SWATH MS: XIC
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Targeted Analysis
Targeted Analysis
Targeted Analysis
Targeted Analysis
Individual Steps
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OpenSwathDecoyGenerator
mass shift & reverse shuffle & pseudo-reverse
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Lam, H., Deutsch, E. W. & Aebersold, R. Artificial decoy spectral libraries for false discovery rate estimation in spectral library searching in proteomics. J. Proteome Res. 9, 605–610 (2010).
OpenSwathRTNormalizer
Outlier Corrected
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OpenSwathChromatogramExtractor
SWATH MS2 Map SWATH XIC
16default extraction window: 0.05 Th
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OpenSwathAnalyzer
Performance assessmentResults
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Gold Standard Dataset
Sample• 422 stable isotope-labeled
peptides• 10x dilution series (0.023 –
11.8 fmol/µL)• Water, Yeast and Human
proteome background• Measured in triplicates• total 90 injections /
472 GB compressed mzML
Manual Analysis• Spectral Library of 349
peptides• Manual analysis using
Skyline:– 32 310
chromatograms analyzed
– 19 416 true peaks annotated
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Performance Assessment: Identification
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AUC: 0.9
Performance Assessment: Identification
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Performance Assessment: Quantification
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Performance Assessment: Quantification
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Next stepsResults
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Alignment of multiple runs
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Results
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Application to a complex microbial sample
Results
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Streptococcus pyogenes: Proteome coverage
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Streptococcus pyogenes: Differential regulation
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14-3-3B interaction dynamics
Results
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– AP-MS of 14-3-3B after IGF-1 stimulation (HEK293)
– 27 samples (time course of 6 samples + 3 controls)
– Assay library with 180'000+ fragment ions
– Quantification of nearly 2000 proteins (550+ high confidence interactions)
* Performed by Dr. Ben Collins. Collins et al., Nature Methods. accepted.31
14-3-3B Interaction dynamics*
● Targeted quantification of 550+ high confidence 14-3-3B interacting proteins across all 27 samples
● Protein quantification over large dynamic range
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14-3-3B Protein Interaction Network
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14-3-3B Protein Interaction Network● Targeted quantification of 550+ high confidence 14-3-3B interacting
proteins across all 27 samples● Protein quantification over large dynamic range
Conclusions
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Conclusion
● Automated targeted analysis of DIA-data provides high reproducibility and high throughput
● OpenSWATH (www.openswath.org)
High recovery / low error rates (AUC > 0.9)
Open Source code (BSD Licence)
Open Formats support (mzML & TraML)
Integration since OpenMS 1.10
Available bindings to Python● Successful applications in bacteria, lower eukaryotes
(not shown) and human cell lines35
Röst and Rosenberger et al. Nature Biotechnology. Accepted.
Acknowledgements
• Supervision– Lars Malmström– Ruedi Aebersold
• OpenSWATH & OpenMS
– Witold Wolski– Hendrik Weisser
• OpenSWATH & ALF– Christina Ludwig– Olga Schubert
• Assays– Samuel Bader– Ben Collins
• MS & Data– Pedro Navarro– Ludovic Gillet– Nathalie
Selevsek
• OpenMS Developers
37Aebersold, Malmström & Kunszt groups
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