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.human leukocyte antigen class I | electron-transfer dissociation | major histocompatibility complex | phosphorylation | binding motif C lass I molecules of the human leukocyte antigen (HLA) complex present short peptides, typically 8-11 aa in length at the cell surface, for scrutiny by the immune system (1). These peptide fragments are generated in the cytoplasm by proteasomal degradation of source proteins, translocated into the endoplasmic reticulum (ER) and subjected to N-terminal trimming to a size that is suitable for loading onto the HLA (2). Loading is governed by physicochemical binding motifs typical for each HLA class I allele (3). Depending on the motif required for the HLA class I allele(s) expressed, an ER-residing peptide may become presented or not. Recognition of specific HLA class I peptide complexes by CD8 T lymphocytes on pathogen infected or cancerous cells leads to the activation of a cytotoxic response and the clearance of the diseased cell. The identification of these HLA class I-associated peptides has important consequences for understanding the biology of cells, vaccine design, and tumor immunotherapy (4, 5).Today mass spectrometry (MS) is the core technology for the analysis of HLA class I-presented peptides. These peptides are typically enriched from cell lysates through the affinity purification of HLA class I peptide complexes, released from the HLA by acid elution, and separated by liquid chromatography (LC) before introduction into the mass spectrometer. Identification is commonly accomplished by...

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry*

“…In fact, this method can be used for the isolation of both class I and class II MHC peptides from a range of biological sources such as cell lines, primary tissues and plasma (36,(51)(52)(53). Moreover, up to ϳ90% of immunopurified peptides were reported to be specific for the MHC molecules (51,54).…”

“…For sequence identification, the recorded MS2 spectra are searched against a protein database using commercial and/or open-source automated search engines (69,70). For MHC class I-associated peptides, ϳ10% of the acquired MS2 spectra can be confidently assigned (false discovery rate Ͻ1%) to a peptide sequence (54). The exact proportion of confidently assigned MS2 spectra might be, however, largely laboratory dependent and might depend on the acquisition parameters of the instrument used (e.g.…”

“…However, the effect of enhancing electrospray ionization and sensitivity might depend on the type of emitter source as well as specific parameters such as voltage, temperature, and gas flow. Interestingly, a gain in HLA class I peptide identification was also found by introducing a novel fragmentation method termed "electron-transfer/ higher-energy collision dissociation" (54). With electron-transfer/higher-energy collision dissociation, Mommen et al found that 39% of the acquired MS2 spectra led to high-confidence HLA class I peptide assignments (54).…”

“…Interestingly, a gain in HLA class I peptide identification was also found by introducing a novel fragmentation method termed "electron-transfer/ higher-energy collision dissociation" (54). With electron-transfer/higher-energy collision dissociation, Mommen et al found that 39% of the acquired MS2 spectra led to high-confidence HLA class I peptide assignments (54). This method generates the fragment ions induced by ETH (c/z) and HCD (b/y) and combines them in a single spectrum (74,75).…”

Minimal Information About an Immuno‐Peptidomics Experiment (MIAIPE)

“…An increasing number of reports have highlighted the influence of the applied fragmentation methodology (e.g., CID, HCD, and EThcD) on the depth and features of the identified immunopeptidome. In fact, MS/MS spectrum generation and acquisition is even more important in immunopeptidomics as compared to other sample types due to the very diverse physical chemical properties of these endogenous peptides 25. Thanks to tremendous technological development in the MS/MS field over the last decade, large‐scale MHC‐class I and MHC‐class II immunopeptidome analyses have taken off in recent years,6, 26, 27 thus drastically increasing the availability of immunopeptidome datasets.…”