Chemical Derivatization Strategy for Extending the Identification of MHC Class I Immunopeptides

Abstract

Neoantigen-based therapeutic vaccines have a high potential impact on tumor eradication and patient survival. Mass spectrometry (MS)-based immunopeptidomics has the capacity to identify tumor-associated epitopes and pinpoint mutation-bearing major histocompatibility complex (MHC)-binding peptides. This approach presents several challenges, including the identification of low-abundance peptides. In addition, MHC peptides have much lower MS/MS identification rates than tryptic peptides due to their shorter sequence and lack of basic amino acid at C-termini. In this study, we report the development and application of a novel chemical derivatization strategy that combines the analysis of native, dimethylated, and alkylamidated peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to expand the coverage of the MHC peptidome. The results revealed that dimethylation increases hydrophobicity and ionization efficiency of MHC class I peptides, while alkylamidation significantly improves the fragmentation by producing more y-ions during MS/MS fragmentation. Thus, the combination of dimethylation and alkylamidation enabled the identification of peptides that could not be identified from the analysis of their native form. Using this strategy, we identified 3148 unique MHC I peptides from HCT 116 cell lines, compared to only 1388 peptides identified in their native form. Among these, 10 mutation-bearing peptides were identified with high confidence, indicating that this chemical derivatization strategy is a promising approach for neoantigen discovery in clinical applications.

Identification of MHC Peptides Using Mass Spectrometry for Neoantigen Discovery and Cancer Vaccine Development

“…Our recent work shows that the analysis of native peptides and their two chemical derivatives can significantly extend immunopeptidome coverage (Fig. 2b) (Chen et al, 2018). Purified MAPs were first dimethylated to block the N‐terminal and lysine residues, followed by alkylamidation of carboxyl groups.…”

“…It is not efficient in identifying singly charged MAPs that comprise at least 30% of the total immunopeptidome. The combination of EThcD with chemical derivatization might is one possible solution, as higher peptide charge states were observed in most MAPs (Chen et al, 2018). Interestingly, HCD in quadrupole‐Obitrap instrument provided higher identification rate (30–40%) compared with the same dissociation method from hybrid LTQ‐Obitrap (Bassani‐Sternberg et al, 2015), which contributed to the higher numbers of unique MAPs identified by quadrupole‐Obitrap instrument.…”

Mass Spectrometry-Based Identification of MHC-Associated Peptides

“…Secondly, they were alkylamidated on the carboxyl groups including C-termini. This modification affects the fragmentation pattern, leading to the generation of more complete y-ion series, increasing the sequence coverage ( Figure 2) [39]. Analysis of the native, dimethylated, and alkylamidated peptides from the same sample enabled the detection of a subset of previously undetected peptides and, thus, led to a substantial increase in immunopeptidome coverage.…”

“…(A) Scheme of the derivatization and (B) tandem MS spectra of synthetic peptide (SVATITGV) in native, dimethylated, and alkylamidated form. Reprinted with permission from Chen et al[39], copyright year 2018, American Chemical Society.…”

Combination of continuous digestion by peptidase and spectral similarity comparisons for peptide sequencing

“…To facilitate peptide fragmentation, various chemical derivatization strategies [19][20][21][22][23][24][25] had been developed. For instance, Chen used 3-(dimethylamino)-1-propylamine to alkylamidate, the carboxyl groups of major histocompatibility complex-binding peptides and improved the fragmentation by producing more y-ions under higher energy collisional dissociation [24]. Frey et al appended tertiary or quaternary amines to peptide carboxyl groups generating more highly charged peptide ions that improved peptide fragmentation [25].…”