Doubling down on phosphorylation as a variable peptide modification.

Some mass spectrometrists believe that searching for variable PTMs like phosphorylation of serine or threonine when using database-search algorithms to interpret peptide tandem mass spectra will increase false-positive matching. The basis for this is the premise that the algorithm compares a spectrum to both a nonphosphorylated peptide candidate and a phosphorylated candidate, which is double the number of candidates compared to a search with no possible phosphorylation. Hence, if the search space doubles, false-positive matching could increase accordingly as the algorithm considers more candidates to which false matches could be made. In this study, it is shown that the search for variable phosphoserine and phosphothreonine modifications does not always double the search space or unduly impinge upon the FDR. A breakdown of how one popular database-search algorithm deals with variable phosphorylation is presented. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.