MS/MS and MS3 spectra of one previously known phospho-peptide originating from Stathmin 1 are shown in Figure 4. A previously unreported phosphorylation site originating from scaffold attachment factor B2 is shown in Figure 5. Scaffold attachment factor B2 has been reported to suppress estrogen receptors and might have an important role in breast cancer (4).
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)
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.
To increase the number of identified phosphopeptides, a greater amount of starting material would be needed (5), and possibly another chromatographic enrichment step specific for phosphopeptides, for example using titanium oxide media (6).
1. Ficarro, S. B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).
2. Application note: Highly sensitive phosphopeptide analysis using Ettan MDLC and a linear ion trap mass spectrometer, GE Healthcare,