Prophossi Peptide-Spectrum Match AnalysisProPhosSI takes a putative peptide match and the associated MS/MS spectrum and examines it according to the following protocol:
- A theoretical spectrum is generated for the putative peptide match
- Observed ions over a user defined relative intensity threshold are matched to theoretical ions with a user defined match threshold
- Analytical rules based on the expertise of experienced mass spectrometrists are applied to the ion matches
- The results of these rules are combined to provide an analytical result
Parent ions presentParent Ions should lose the labile phosphates from the S and T residues as the first stage in fragmentation. A positive test occurs when parent ions corresponding to the des-phospho peptide are observed in the spectrum.
Three -98 Ions presentParent Ions should lose the labile phosphates from the S and T residues as the first stage in fragmentation. Subsequent fragment ions should then be des-phospho. A positive test occurs when at least three b or y series ions corresponding to fragments of the des-phospho peptide are observed in the spectrum.
Unique -98 transitions presentSerine and threonine residues lose phosphate (98Da). There should be peaks in the y or b series spanning the phosphorylation site and indicating the phosphate loss. No other S/T residues should be present in the spanned region. WARNING: For peptides like -S-X-S-X-S-, if both singly phosphorylated S1 and S3 are present, this test can report a false positive for S2.
Four Sequential b or y ionsObserving a sequence of b or y ions gives confidence that the fragmentation spectrum is high quality and corresponds to the peptide matched by Mascot.
Five of six sequential ions presentFragmentation of peptides produces two main series of ions, b and y. The difference between adjacent ions in the series corresponds to the mass of the amino acid residues. A sequence of peaks corresponding to these fragments can therefore be used to identify the sequence. This rule allows for one missing peak in a series of six.
Proline directed fragmentation patternFragmentation around proline gives a very distinctive pattern. The N-terminal fragmentation (cleaving the imino bond N-terminal to the residue) should be one of the major peaks in the spectrum. Fragmentation of the amino bond (C-terminal to the residue) is much less common so should be very weak. This test passes if there are no proline or if at least one of the prolines present have the correct fragment intensity distribution.Some residues inhibit cleavage at the imino bond. These are flagged in the results.
PhosphoTyrosine transition presentThe phosphate group on Tyrosine residues is not labile so the mass difference between the series ions either side of the tyrosine should be present.
Six of top ten ions identifiedThe most intense ions in the spectrum should be identified in order to be confident that the spectrum corresponds to that particular peptide. If at least six of the top ten ions are identified then this test passes.
ProPhosSI RulesTo validate a peptide, ProPhosSI requires three -98 ions, either four sequential or five of six b or y ions, an appropriate proline directed fragmentation pattern and at least six of the top ten ions matched to the theoretical spectrum.
To localise the phosphorylation site, specific and unambiguous phosphotyrosine or -98 transitions are required.