Thirty of the collected fractions were further analyzed by LC-MS using the neutral loss function. Phosphopeptides were found in one-third of the analyzed SCX fractions as shown in Figure 2. They mainly eluted early in the salt gradient due to their reduced charge state (4). SCX can therefore be used as a phosphopeptide enrichment strategy.
An ion chromatogram and the MS3 events (i.e. where a neutral loss was detected) from one of the fractions are shown in Figure 3.
The phosphopeptides were found and the phosphorylation sites identified by TurboSEQUEST database searches on all MS3 spectra. The results were confirmed manually by studying the raw spectra. It was important to confirm that the charge state of the peptide was correct, that the neutral loss ion dominated the MS/MS spectrum (Fig 4), and that the sequence data was of high quality.
Database searches were then performed on all MS/MS spectra, and the results were used to confirm the MS/MS searches and to find tyrosine phosphorylations. Phospho-tyrosine does not lose phosphoric acid during collision in the ion trap; therefore, sequence data from MS/MS was used to find these phosphorylations (+80). Some possible tyrosine phosphorylations were identified but need further evaluation. Tyrosine-phosphorylated proteins exist in very low abundance in cells and probably fall below the detection limit of the method.
In total, 60 phosphorylated peptides were found originating from 50 proteins. Some of the identified phosphorylation sites are shown in Table 1. The proteins presented in the table are al