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!
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 MS1 • 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) • Advantages • Disadvantages • HCD (Higher energy Collisional Dissociation) New fragmentation technique High-high strategy
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
HCD spectra vs CID spectra HCD spectra generated by LTQ-OrbitrapVelos 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).
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?
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?