The Human Leukocyte Antigen–presented Ligandome of B Lymphocytes

Abstract

The presented HLA class I ligands are the products of the intracellular processing machinery, with its continuous cycle of protein synthesis and degradation (3). Much is known about the proteins involved in antigen processing, but high fidelity ligand/epitope predictions are at present not possible. The discovery of additional involved enzymes (3, 4) and the exciting discovery of peptide splicing (5) have shown that antigen processing is even more complex than was previously thought. Moreover, gene expression studies have shown many nonstandard, unexpected protein products, including the production of antigens derived from aberrant protein fragments as a result of expression in alternative reading frames (6). Several studies report the identification of HLA ligands (7-10). Many results have been collected and discussed in a recent review on the large-scale analysis of HLA class I ligands (11). Collectively, these reports illustrate the need for in-depth elucidation of the HLA ligandome.Elucidation of T cell epitopes has traditionally been achieved with the use of a forward immunological approach, as pioneered by Hunt and coworkers (12,13). In this approach, the cognate peptide of T cells with the appropriate activity profile is elucidated via repeated rounds of chromatographic separation in combination with T cell recognition assays. Because T cells are not always available from the start, reverse immunological approaches (14 -17) have been developed to predict T cell epitopes through a combination of bioinformatics and in vitro proteasome digests. Predicted epitopes are synthesized and tested for their capability to activate T cells. The main disadvantage of this approach is that less than 0.1% of the peptides that survive intracellular processing are presented on HLA class I molecules (3).Therefore, we developed a large-scale peptidomics approach that is a reverse immunology approach based not on From the ‡Department of Immunohematology and Blood Transfusion,

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

“…Comparison of the complete dataset with recent large-scale studies revealed that 81% of the peptides have not been reported before (SI Appendix, Table S2) (16)(17)(18). Remarkably, the unique peptides identified in this study have distinct sequence characteristics compared with literature data (SI Appendix, Fig S4).…”

“…S9). Comparison with previous studies revealed that 16 HLA-B7-associated phosphopeptides had been reported previously (16,24,26), whereas a total of 24 phosphorylation sites are annotated in the Uniprot protein database (www.uniprot.org/). The correct identification of selected phosphopeptides could be nicely confirmed by the spectra we generated from their synthetic peptide counterparts (SI Appendix, Fig.…”

“…S6). Moreover, for the peptides related to these proteins, no relation was found between peptide intensity, allele specificity, or predicted binding strength, confirming that the final level of presentation depends on many factors [e.g., proteolytic activity, transporter-associated with antigen processing (TAP) transport efficiency] (16,20).…”

Minimal Information About an Immuno‐Peptidomics Experiment (MIAIPE)

“…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.…”

“…have reviewed the three main data acquisition concepts in MS that have been applied in immunopeptidomics,2 commonly used in other proteomics MS workflows. These include: 1) data‐dependent analysis (DDA) which is the widely used strategy for so‐called “discovery experiments” where the researcher is interested in profiling the repertoire of pMHC from a given sample5, 6; 2) selected reaction monitoring (SRM), also referred to as multiple reaction monitoring (MRM), targeted analyses whereby ion signatures from both the peptide precursor and its fragmentation product ions are monitored, and the area under the curve or peak height can be used for quantification purposes; this method ensures a higher degree of selectivity than a DDA experiment, but at the expense of the experimental depth and throughput7; and 3) data independent analysis (DIA) strategies, where all peptides within a defined mass‐to‐charge ( m/z ) window are subjected to fragmentation and recording of their combined MS/MS spectra; the precursor isolation window sequentially marches up the full m/z range and this analysis is repeated again and again during chromatographic elution 8. This less mature technique promises excellent performance for both discovery and quantitative analyses, especially with regard to reproducibility and more accurate label‐free quantification 9…”