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