Targeted Proteomics Environment

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Presentation transcript:

Targeted Proteomics Environment Targeted Proteomics Quantitative Analysis of Data Independent Acquisition MS/MS in Skyline Brendan MacLean; Jarrett Egertson; Donald M. Marsh; Michael J. MacCoss

2010 Multi-Instrument SRM Tool for NCI CPTAC Verification Working Group AB SCIEX 4000 Q Trap Thermo- Scientific TSQ Ultra 4000 Q Trap data from Jeff Whiteaker TSQ data from MacCoss Lab Unrelated to any VWG study Kessner, D. et al. Bioinformatics. 2008.

2010 Graphic Display of Information Broad Institute Study 7 data

2010 Skyline Adoption (>1500 installations)

Quantitative Proteomics Spectrum-based Spectral counting Isobaric tags Chromatography-based SRM MS1 chromatogram extraction Targeted MS/MS Data independent acquisition (DIA)

Skyline MS1 Full-Scan Settings Tutorial

Skyline MS1 Filtering Data Schilling, B. et al. Mol. Cell. Prot. 2012.

Targeted MS/MS MS/MS scans intensity time (scan #) 2 m/z isolation m/z Ion Source CID Q1 / Trap Mass Analyzer 2 m/z isolation MS/MS scans time (scan #) intensity m/z 521.27

Skyline Targeted MS/MS Settings Tutorial

Skyline Targeted MS/MS Sherrod, S. et al. J. Prot. Res. 2012.

Skyline Targeted MS/MS Sherrod, S. et al. J. Prot. Res. 2012.

2010 Support Multiple Instrument Vendors SRM Exporting transition lists & native methods Importing native instrument output files AB SCIEX Agilent Technologies Thermo-Scientific Waters

2012 Support Multiple Instrument Vendors Full-Scan Exporting isolation lists & native methods Importing native instrument output files AB SCIEX SWATH™ Agilent Technologies DIA Thermo-Scientific DIA & Multiplexed DIA Waters MSe™

Traditional DIA Isolation Scheme precursor m/z SVEDFMAAMQR Precursor m/z: 616 600 610 616 620 630 640 650 619 VGGNGADYALATK Precursor m/z: 619 10 m/z x Retention time Venable, J.D. et al. Nat. Meth. 2004.

DIA Chromatogram Extraction Mass Analyzer Q1 / Trap CID Ion Source 10 m/z isolation MS/MS scans time (scan #) intensity m/z

DIA Chromatogram Extraction Trade-Offs Sensitivity Dwell / Accumulation time Cycle time Selectivity lost by wide precursor isolation Selectivity gained vs. SRM by narrow product extraction MS/MS scan Gillet, L.C. et al. Mol. Cell. Prot. 2012.

Skyline Agilent DIA Settings (11.5 m/z Extraction Windows)

Agilent DIA Data (Product Ions)

Agilent DIA Data (Precursor Ions)

Skyline AB SCIEX SWATH™ Settings in v1 Skyline AB SCIEX SWATH™ Settings in v1.3 (32 x 25 m/z Extraction Windows) Gillet, L.C. et al. Mol. Cell. Prot. 2012.

AB SCIEX SWATH™ Data Gillet, L.C. et al. Mol. Cell. Prot. 2012.

Skyline Waters MSe™ Settings in v1.3 (50 – 2000 m/z Range)

Waters MSe™ Data

Multiplexed DIA (Q Exactive) in v1.3 Jarrett Egertson – Thursday AM – ThP 571 Scan N-1 100 4 m/z-wide windows = 400 m/z (5 random per scan) Scan N Scan N+1 MSX Acquisition De-Multiplexed Spectra De-Multiplexing Scan N Scan N-1 Scan N+1 m/z Multiplexed Spectra . . . Isolation Window (present = 1; absent = 0) Precursor Isolation x =

Multiplexed DIA Data (before) Jarrett Egertson – Thursday AM – ThP 571

Multiplexed DIA Data (after) Jarrett Egertson – Thursday AM – ThP 571

2012 Skyline Adoption (>10,000 installations) ASMS 2010 2011 Skyline Instances Started Trailing 7 Days Instances 3500 3000 2500 1500 1000 500 2000 Dev 64 Release Dev Release 1.2 Release 1.1 Release 0.7 Release Pre-0.7 Release

Getting Started Freely available & Open Source http://proteome.gs.washington.edu/software/skyline Self-updating web installation (v1.3 coming soon) 2 full-scan filtering tutorials (DIA coming soon) Support board and issues list 8 other tutorials & 3 instructional videos (full-scan video soon) MS1 Full-Scan Filtering Targeted MS/MS Data Independent Acquisition Coming Soon

Acknowledgments: Thermo-Scientific Waters Skyline Team Buck Institute Nick Shulman Vagisha Sharma Barbara Frewen Michael Bereman Buck Institute Birgit Schilling Matthew Rardin Brad Gibson ETH Rudolph Aebersold Ludovic Gillet Christina Ludwig Vanderbilt Matthew Chambers Amy Ham Daniel Liebler Duke Will Thompson Arthur Moseley AB Sciex Fadi Abdi David Cox Christie Hunter Brent Lefebvre Agilent Technologies Christine Miller Joe Roark Pat Perkins Thermo-Scientific Markus Kellmann Andreas Kuehn Vlad Zabrouskov Waters Laurence Firth James Langridge Roy Martin Kieran Neeson Keith Richards WP 407 - Vagisha Sharma A private repository of targeted proteomics assays for Skyline This work is funded by grants from NIH/NIGMS, NIH/NHGRI, Agilent Technologies and Thermo-Fisher Scientific.