1. 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

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

4. MS technologies are evolving

5. 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

6. 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!

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

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

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

10. HCD

11. 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

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

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

14. 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.

15. 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).

16. Comparison of HCD and CID spectra

17. Comparison of HCD and CID spectra

18. Mass accuracy

19. HCD vs CID for high-high strategy

20. 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?

21. Sensitivity issue

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

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

24. 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?