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

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Presentation transcript:

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

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

MS technologies are evolving

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

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

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

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

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

HCD

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-Orbitrap Velos

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

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

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.

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).

Comparison of HCD and CID spectra

Comparison of HCD and CID spectra

Mass accuracy

HCD vs CID for high-high strategy

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?

Sensitivity issue

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

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

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?