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Although database search tools originally developed for shotgun proteome have been widely used in immunopeptidomic mass spectrometry identifications, they have been reported to achieve undesirably low sensitivities or high false positive rates as a result of the hugely inflated search space caused by the lack of specific enzymic digestions in immunopeptidome. To overcome such a problem, we developed a motif-guided immunopeptidome database building tool named IntroSpect, which is designed to first learn the peptide motifs from high confidence hits in the initial search, and then build a targeted database for refined search. Evaluated on 18 representative HLA class I datasets, IntroSpect can improve the sensitivity by an average of 76%, compared to conventional searches with unspecific digestions, while maintaining a very high level of accuracy (~96%), as confirmed by synthetic validation experiments. A distinct advantage of IntroSpect is that it does not depend on any external HLA data, so that it performs equally well on both well-studied and poorly-studied HLA types, unlike the previously developed method SpectMHC. We have also designed IntroSpect to keep a global FDR that can be conveniently controlled, similar to a conventional database search. Finally, we demonstrate the practical value of IntroSpect by discovering neoepitopes from MS data directly, an important application in cancer immunotherapies. IntroSpect is freely available to download and use.

biomolecules

IntroSpect: Motif-Guided Immunopeptidome Database Building Tool to Improve the Sensitivity of HLA I Binding Peptide Identification by Mass Spectrometry