Targeted Proteomics Environment
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1 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
2 2010 Multi-Instrument SRM Tool for NCI CPTAC Verification Working Group AB SCIEX 4000 Q Trap Thermo- Scientific TSQ Ultra Kessner, D. et al. Bioinformatics
3 2010 Graphic Display of Information
4 Instances Per Week 2010 Skyline Adoption (>1500 installations) 1200 Skyline Average Usage Per Week Daily 0.6 Release 0.5 Release 0.5 Preview 0.2 Release 0
5 Quantitative Proteomics Spectrum-based Spectral counting Isobaric tags Chromatography-based SRM MS1 chromatogram extraction Targeted MS/MS Data independent acquisition (DIA)
6 Skyline MS1 Full-Scan Settings
7 Skyline MS1 Filtering Data Schilling, B. et al. Mol. Cell. Prot
8 Targeted MS/MS Ion Source Q1 / Trap CID Mass Analyzer 2 m/z isolation MS/MS scans
9 Skyline Targeted MS/MS Settings
10 Skyline Targeted MS/MS Sherrod, S. et al. J. Prot. Res
11 Skyline Targeted MS/MS Sherrod, S. et al. J. Prot. Res
12 2010 Support Multiple Instrument Vendors SRM Exporting transition lists & native methods Importing native instrument output files AB SCIEX Agilent Technologies Thermo-Scientific Waters
13 2012 Support Multiple Instrument Vendors Full-Scan Exporting isolation lists & native methods Importing native instrument output files AB SCIEX Agilent Technologies Thermo-Scientific Waters SWATH DIA DIA & Multiplexed DIA MSe
14 Traditional DIA Isolation Scheme SVEDFMAAMQR Precursor m/z: 616 VGGNGADYALATK Precursor m/z: 619 x x x x 10 m/z precursor m/z Retention time Venable, J.D. et al. Nat. Meth
15 DIA Chromatogram Extraction Q1 / Trap CID Mass Analyzer Ion Source 10 m/z isolation MS/MS scans
16 DIA High-Res. Chromatogram Extraction 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
17 Skyline Agilent DIA Settings (11.5 m/z Extraction Windows)
18 Agilent DIA Data (Product Ions)
19 Agilent DIA Data (Precursor Ions)
20 Skyline AB SCIEX SWATH Settings in v1.3 (32 x 25 m/z Extraction Windows) Gillet, L.C. et al. Mol. Cell. Prot
21 AB SCIEX SWATH Data Gillet, L.C. et al. Mol. Cell. Prot
22 Skyline Waters MSe Settings in v1.3 ( m/z Range)
23 Waters MSe Data
24 De-Multiplexing Isolation Window Acquisition Multiplexed DIA (Q Exactive) in v1.3 Jarrett Egertson Thursday AM ThP 571 MSX Scan N-1 Scan N Scan N m/z-wide windows = 400 m/z (5 random per scan) Multiplexed Spectra Precursor Isolation De-Multiplexed Spectra Scan N-1 Scan N Scan N+1 m/z =... Scan N-1 Scan N Scan N+1... Isolation Window (present = 1; absent = 0) x m/z
25 Multiplexed DIA Data (before) Jarrett Egertson Thursday AM ThP 571
26 Multiplexed DIA Data (after) Jarrett Egertson Thursday AM ThP 571
27 Instances 2012 Skyline Adoption (>10,000 installations) 3500 Skyline Instances Started Trailing 7 Days ASMS 2011 Dev 64 Release 2000 Dev Release 1500 ASMS Release 1.1 Release Release Pre-0.7 Release 500 0
28 Getting Started Freely available & Open Source Self-updating web installation (v1.3 coming soon) 2 full-scan filtering tutorials (DIA coming soon) MS1 Full-Scan Filtering Targeted MS/MS Data Independent Acquisition Support board and issues list 8 other tutorials & 3 instructional videos (full-scan video soon)
29 Acknowledgments: Skyline Team Nick Shulman Vagisha Sharma Barbara Frewen Michael Bereman Buck Institute ETH Birgit Schilling Matthew Rardin Brad Gibson 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 This work is funded by grants from NIH/NIGMS, NIH/NHGRI, Agilent Technologies and Thermo-Fisher Scientific. Thermo-Scientific Markus Kellmann Andreas Kuehn Vlad Zabrouskov Waters Laurence Firth James Langridge Roy Martin Kieran Neeson Keith Richards WP Vagisha Sharma A private repository of targeted proteomics assays for Skyline
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