Improving MHC-I ligand identification by incorporating targeted searches of mass spectrometry data

Effective immunotherapies rely on specific activation of immune cells. Class I major histocompatibility complex (MHC-I) bound peptide ligands play a major role in dictating the specificity and activation of CD8+ T cells and hence are important in developing T cell-based immunotherapies. Mass spectrometry-based approaches are most commonly used for identifying these MHC-bound peptides, wherein the MS/MS spectra are compared against a reference proteome database. Unfortunately, the effectiveness of matching the immunopeptide MS/MS spectra to a reference proteome database is hindered by inflated search spaces attributed to a lack of enzyme restriction in searches. These large search spaces limit the efficiency with which MHC-I peptides are identified. Here, we describe the implementation of a targeted database search approach and accompanying tool, SpectMHC, that is based on a priori-predicted MHC-I peptides. We have previously shown that this targeted search strategy improved peptide identifications for both mouse and human MHC ligands by greater than two-fold and is superior to traditional "no enzyme" search of reference proteomes (Murphy et al. J Res Proteome 16:1806-1816, 2017).