Table 1.

Analysis Criteria for automated validation of PSMs

Prefilter criteria
P1	Forward hit	Only hits against forward, non-redundant sequences are selected
P2	Mass accuracy	Only hits within 0.1 Da (or 0.1 + 1 Da) of the parent ion m/z are selected.
P3	Phospho-PTM	Only hits containing a putative Phospho PTM are selected
P4	Within 20 points	Only hits which are within 20 MASCOT score points of the top ranked hit for that query are selected.
P5	Over FDR threshold	Only peptides with a MASCOT score over the calculated FDR 1% threshold are selected.
Validation Criteria—Phosphopeptide assignment
1	4 in a row	At least four sequential y- or b-series ions are present. This indicates good coverage of the peptide.
2	5 of 6	5 out of 6 sequential b- or y-series ions are present. This indicates good coverage of the peptide.
3	3 desphospho ions	At least three y- or b-series ions with a phosphate loss are present. The phosphate ester bond tends to be more labile than the peptide bond.
4	Proline-directed fragmentation	The imino bond to the N-terminal side of a proline residue is particularly labile. If the sequence contains a Proline residue then at least one of the imino bonds should give a fragment ion with at least 50% maximum intensity and much stronger than the relatively weakly cleaved amide bond C-terminal to Proline.
5	6 of top 10 ions	6 of the 10 most intense ions should be assigned to y- or b-series ions.
Validation criteria—phosphosite assignment
6	Phosphate transitions	To assign the site specifically, at least one ion unique to that peptide species must be observed. This is aided by the high rate of phosphate loss from pSer and pThr residues.
7	PhosphoTyrosine	Mass differences corresponding to pTyr should be observed between identified peaks.