PhosphoScore:: An open-source phosphorylation site assignment tool for MSn data

Correct phosphorylation site assignment is a critical aspect of phosphoproteomic analysis. Large-scale phosphopeptide data sets that are generated through liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis often contain hundreds or thousands of phosphorylation sites that require validation. To this end, we have created PhosphoScore, an open-source assignment program that is compatible with phosphopeptide data from multiple MS levels (MSn). The algorithm takes into account both the match quality and normalized intensity of observed spectral peaks compared to a theoretical spectrum. PhosphoScore produced >95% correct MS2 assignments from known synthetic data, > 98% agreement with an established MS2 assignment algorithm (Ascore), and >92% agreement with visual inspection of MS3 and MS4 spectra.