Multiaspect Examinations of Possible Alternative Mappings of Identified Variant Peptides: A Case Study on the HEK293 Cell Line

Adopting proteogenomics approach to validatesingle nucleotide variation events by identifying correspondingsingle amino acid variant peptides from mass spectrometry (MS)-based proteomics data facilitates translational and clinical research.Although variant peptides are usually identified from MS data witha stringent false discovery rate (FDR), FDR control could fail toeliminate dubious results caused by several issues; thus,postexamination to eliminate dubious results is required. However,comprehensive postexaminations of identification results are stilllacking. Therefore, we propose a framework of three bottom-uplevels, peptide-spectrum match, peptide, and variant event levels,that consists of rigorous 11-aspect examinations from the MSperspective to further confirm the reliability of variant events. As aproof of concept and showing feasibility, we demonstrate 11 examinations on the identified variant peptides from an HEK293 cellline data set, where various database search strategies were applied to maximize the number of identified variant PSMs with an FDR<1% for postexaminations. The results showed that only FDR criterion is insufficient to validate identified variant peptides and the11 postexaminations can reveal low-confidence variant events detected by shotgun proteomics experiments. Therefore, we suggestthat postexaminations of identified variant events based on the proposed framework are necessary for proteogenomics studies