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Sangtae Kim Ph.D. candidate University of California, San Diego

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  1. UCSD Mass Spectrometry Journal Club Feasibility of Large-Scale Phosphoproteomics with Higher Energy Collisional Dissociation Fragmentation Sangtae Kim Ph.D. candidate University of California, San Diego 11/12/2010 Center for Computational Mass Spectrometry

  2. What is it about? • Can we use the HCD technology for large-scale phosphoproteomics studies? Yes!

  3. MS technologies are evolving

  4. Why are people crazy about high-precision spectra? • High-precision MS1? • High-precision MS2? • Unambiguous precursor charge determination • Decrease the search space • - Unambiguous fragment ion charge determination • - Better separation of signal/noise

  5. Strategies to generate mass spectra • High-low strategy • High-precision MS1 • Low-precision MS2 • High-high strategy • High-precision MS1 • High-precision MS2 High-low strategy is preferred!

  6. How much do we benefit from high-precision MS/MS spectra? MS-GFDB search results with QTOF dataset from Agilent

  7. Why are people still using ion-trap for MS2? • Cheap • Sensitive • Fast

  8. Fragmentation technologies • CID (Collision Induced Dissociation) • Advantages • Disadvantages • ETD (Electron Transfer Dissociation) • Advantages • Disadvantages • HCD (Higher energy Collisional Dissociation) New fragmentation technique High-high strategy

  9. HCD

  10. HCD features • Requires LTQ-Orbitrap • High-resolution, high-accuracy MS2 • No loss of low mass ions • Lower sensitivity! Immonium ions are detectable! - not so bad with LTQ-OrbitrapVelos

  11. HCD spectra vs CID spectra HCD spectra generated by LTQ-OrbitrapVelos machine is much better than this (10-fold more ion current)

  12. Claim • High-high strategy using HCD is as good as or better than high-low strategy! • For phosphosproteome analysis

  13. Dataset • HeLa S3 cells • Enrichment of phosphopeptides by TiO2 beads • Mascot 2.2 • Precursor mass tolerance 7ppm • Fragment mass tolerance 0.02Da or 0.5Da • C+Carbamidomethylation fixed • N-acetyl Prot, Ox Met, Phospho STY • 2 missed cleavages • 1% FDR at three levels (site, peptide, protein) • For HCD/CID, up to most 10/20 peaks were selected for MS2 fragmentation.

  14. Results • With the high-high strategy with HCD, they identified 9668 (class I) phosphorylation sites (16559 total). • With the high-low strategy with CID (pseudo MS3 mode), they identified 9016 (class I) phosphorylation sites (11893 total).

  15. Comparison of HCD and CID spectra

  16. Comparison of HCD and CID spectra

  17. Mass accuracy

  18. HCD vs CID for high-high strategy

  19. Sensitivity issue • HCD requires more ions to generate good quality spectra (ion target value 30,000 for HCD vs 5,000 for CID). • For many spectra, the target-value was not reached because of the injection time limit (150ms). • Is is fine?

  20. Sensitivity issue

  21. Are identifications from HCD spectra different from those from CID spectra?

  22. Conclusion • High-high strategy using HCD is as good as or better than high-low strategy using CID.

  23. Criticisms • All experimental results are based on Mascot search • Mascot does not fully benefit from high-accuracy (limit 0.25Da) • HCD results are better than presented?