Sangtae Kim Ph.D. candidate University of California, San Diego
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Transcript of Sangtae Kim Ph.D. candidate University of California, San Diego
Feasibility of Large-Scale Phosphoproteomics with Higher Energy Collisional Dissociation Fragmentation
Sangtae Kim
Ph.D. candidateUniversity of California, San Diego
Center forComputationalMassSpectrometry
UCSD Mass Spectrometry Journal Club
11/12/2010
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
What is it about?
• Can we use the HCD technology for large-scale phosphoproteomics studies?
Yes!
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
MS technologies are evolving
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
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
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
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!
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
How much do we benefit from high-precision MS/MS spectra?
MS-GFDB search results with QTOF dataset from Agilent
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Why are people still using ion-trap for MS2?
• Cheap
• Sensitive
• Fast
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Fragmentation technologies
• CID (Collision Induced Dissociation)– Advantages– Disadvantages
• ETD (Electron Transfer Dissociation)– Advantages– Disadvantages
• HCD (Higher energy Collisional Dissociation)New fragmentation techniqueHigh-high strategy
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
HCD features
• Requires LTQ-Orbitrap• High-resolution, high-accuracy MS2• No loss of low mass ions
• Lower sensitivity! - not so bad with LTQ-Orbitrap Velos
Immonium ions are detectable!
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
HCD spectra vs CID spectra
HCD spectra generated by LTQ-Orbitrap Velos machine is much better than this (10-fold more ion current)
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Claim
• High-high strategy using HCD is as good as or better than high-low strategy!
– For phosphosproteome analysis
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
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.
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
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).
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Comparison of HCD and CID spectra
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Comparison of HCD and CID spectra
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Mass accuracy
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
HCD vs CID for high-high strategy
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
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?
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Sensitivity issue
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Are identifications from HCD spectra different from those from CID spectra?
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
Conclusion
• High-high strategy using HCD is as good as or better than high-low strategy using CID.
Center forComputationalMassSpectrometryhttp://proteomics.ucsd.edu
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?