P-VIS: internally-controlled validation of mass spectrometry-based peptide identifications.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an increasingly powerful tool for studying proteins in the context of disease. As technological advances in instrumentation and data analysis have enabled deeper profiling of proteomes and peptidomes, the need for a rigorous, standardized approach to validate individual peptide-spectrum matches (PSMs) has emerged. To address this need, we developed a novel and broadly-applicable workflow: PSM Validation with Internal Standards (P-VIS). In this approach, the fragmentation spectrum and chromatographic retention time of a peptide within a biological sample are compared with those of a synthetic version of the putative peptide sequence match. Similarity measurements obtained for a panel of internal standard peptides are then used to calculate a prediction interval for valid matches. If the observed degree of similarity between the biological and the synthetic peptide falls within this prediction interval, the match is considered valid. P-VIS enables systematic and objective assessment of the validity of individual PSMs, providing a measurable degree of confidence when identifying peptides by mass spectrometry.