Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD)

Abstract

The identification of peptides presented by human leukocyte antigen (HLA) class I is tremendously important for the understanding of antigen presentation mechanisms under healthy or diseased conditions. Currently, mass spectrometry-based methods represent the best methodology for the identification of HLA class I-associated peptides. However, the HLA class I peptide repertoire remains largely unexplored because the variable nature of endogenous peptides represents difficulties in conventional peptide fragmentation technology. Here, we substantially enhanced (about threefold) the identification success rate of peptides presented by HLA class I using combined electron-transfer/higher-energy collision dissociation (EThcD), reporting over 12,000 high-confident (false discovery rate <1%) peptides from a single human B-cell line. The direct importance of such an unprecedented large dataset is highlighted by the discovery of unique features in antigen presentation. The observation that a substantial part of proteins is sampled across different HLA alleles, and the common occurrence of HLA class I nested sets, suggest that the constraints of HLA class I to comprehensively present the health states of cells are not as tight as previously thought. Our dataset contains a substantial set of peptides bearing a variety of posttranslational modifications presented with marked allele-specific differences. We propose that EThcD should become the method of choice in analyzing HLA class I-presented peptides.

Keywords: binding motif; electron-transfer dissociation; human leukocyte antigen class I; major histocompatibility complex; phosphorylation.

Fig. 1.

Peptide fragmentation by ETD, HCD, and EThcD. Illustrative MS/MS spectra of the HLA-B27-associated peptide YRAPELLL upon fragmentation by (A) ETD, (B) HCD, and (C) EThcD. The observed and/or assignable c/z and b/y fragment ions are indicated above and below the peptide sequence. MS/MS of the synthetic peptide counterpart are provided in SI Appendix, Fig. S11.

Fig. 2.

Performance characteristics of peptide fragmentation techniques for HLA class I-associated peptides. Shown are identification results and binding motifs of peptides presented by different HLA class I molecules on the surface of GR cells. (A) The number of unique 8- to 14-mer peptides identified by the 1D and 2D peptide separation strategy, assigned to HLA-A1, -A3, -B7, and -B27 molecules, or not assigned to any of the above (NA). The 1D strategy comprised consecutive single LC-MS/MS analysis runs, using either EThcD (black), sequential CID/HCD (red), CID (yellow), HCD (green), or ETD (blue). The 2D strategy included the analysis of SCX fractions by LC-MS/MS using EThcD (black) or sequential CID/HCD (red). (B) Sequence logos showing that peptides are bound to a particular HLA molecule through a distinct binding motif, typically by anchor residues on position P2 (P3) and the C-terminal PΩ residue. (C) Bar diagrams depicting the peptide fraction identified by EThcD (black), the overlap between the methods (gray), and uniquely identified by CID/HCD (red), CID (yellow), HCD (green), or ETD (blue). (D) The number of unique peptides identified by the different peptide fragmentation methods (color coded) in the 1D and 2D strategy. The line graph depicts the cumulative number of unique HLA class I-associated peptides (left y axis) and the peptide identification fraction (right y axis).