( In total 60 phosphorylated peptides we...)Sensitive identification of phosphopeptides in brain tissue using Ettan MDLC and Finnigan LTQ

The strategy for analyzing phosphopeptides confidently is summarized here:
1. 2D-LC (SCX/RPC)
2. MS³ on all peptides that show neutral loss of phosphoric acid
3. TurboSEQUEST searches on all MS³ spectra (-18@ST)
4. Manual confirmation of charge state and that neutral loss dominates MS/MS spectra
5. Further confirmation by MS/MS searches (+80@STY)

The phosphopeptides were found using database searches on all MS³ spectra by TurboSEQUEST software, and were further confirmed manually by studying the raw spectral data. It was important to confirm that the charge state of the peptide was correct, that the neutral loss dominated the MS/MS spectrum (Fig 3), and that the sequence data was of high quality.

Database searches were then performed on all MS/MS spectra. The results were used to confirm the MS/MS searches and to find tyrosine phosphorylations. Phosphorylated tyrosine does not lose phosphoric acid during collision in the ion trap; the sequence data from MS/MS was therefore used to find these phosphorylations. A few tyrosine phosphorylations were found.

Conclusions
To confidently assign phosphopeptides in a complex mixture such as a tryptic digest of brain tissue, two-dimensional separations are needed. 2D-LC separated the peptides with high resolution, and the neutral loss MS detection was very selective for phosphopeptides. Care had to be taken when interpreting the data to avoid false positives from the database searches.

References
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