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

http://proteomics.ucsd.edu/


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 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 techniqueHigh-high strategy



HCD



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!



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?


Documents

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