catalyzed peptide splicing.Abbreviations: electron-transfer higher-energy collision dissociation (EThcD); higher-energy collision dissociation (HCD); false discovery rate (FDR); HLA class I (HLA-I); lymphoblastoid cell line (LCL); mass spectrometry (MS); molecular weight (MW); proteasome-catalyzed peptide splicing (PCPS). 2The proteasome generates the epitopes presented on HLA class I molecules that elicit CD8 + T cell responses. While reports of proteasome-generated spliced epitopes exist, they have been regarded as rare events. Here, however, we show that the proteasome-generated spliced peptide pool accounts for one third of the entire HLA class I immunopeptidome in terms of diversity and one fourth in terms of abundance. They also represent a unique set of antigens, possessing particular and distinguishing features. We validated this observation using a range of complementary experimental and bioinformatics approaches, and multiple cell types. The widespread appearance and abundance of proteasome-catalyzed peptide splicing events will inform immunobiology and autoimmunity theories, and may provide a previously untapped source of epitopes for uses in vaccines and cancer immunotherapy.3The presentation of epitopes on the cell surface is a key mechanism by which organisms identify the presence of pathogens, metabolic malfunctioning, or tumors. The HLA class I (HLA-I) immunopeptidome, i.e. the epitopes allocated onto the HLA-I molecules, impinges upon the CD8 + T cell repertoire and the cell-mediated immune response (1). HLA-I immunopeptidomes are usually investigated by sequence identification of peptides eluted from HLA-I molecules using LC-tandem mass spectrometry (MS) (Fig. S1). The key step for the transformation of a protein into HLA-Irestricted epitopes is usually processing by the proteasome (1), which cuts proteins into peptides;alternatively, the proteasome can also cut and paste peptide sequences, thereby releasing peptide antigens that do not correspond to the original protein sequence (2) (Fig. S2). This proteasomecatalyzed peptide splicing (PCPS) has long been considered to occur only very rarely; partly this has been because the screening of the HLA-I immunopeptidome for proteasome-generated spliced peptides was impeded by methodological challenges.To overcome these problems we developed an analytical strategy, which accounts for recent discoveries underpinning the PCPS mechanism, and which can handle the vast proteome-wide human spliced peptide database (Fig. S3). With this strategy we initially analyzed the HLA-I-eluted immunopeptidome of the GR lymphoblastoid cell line (GR-LCL) adopting, for a deeper coverage of the immunopeptidome, a 2D peptide pre-fractionation strategy, followed by a hybrid peptide fragmentation method, electron-transfer higher-energy collision dissociation (EThcD) for peptide identification (3, 4) ( Fig. S1), supplemented by an adapted target-decoy approach (Fig. S4). Our analysis led to the identification of 6592 non-spliced and 3417 spliced 9-12mer peptides ( Table S1)....

Comprehensive knowledge of the human leukocyte antigen (HLA) class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their purification for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential immuno-affinity purification of HLA-I and -II peptides from up to 96 samples in a plate format, suitable for both cell lines and tissues. Our methodology drastically reduces sample-handling and can be completed within five hours. We challenged our methodology by extracting HLA peptides from multiple replicates of tissues (n = 7) and cell lines (n = 21, 108 cells per replicate), which resulted in unprecedented depth, sensitivity and high reproducibility (Pearson correlations up to 0.98 and 0.97 for HLA-I and HLA-II). Because of the method's achieved sensitivity, even single measurements of peptides purified from 107 B-cells resulted in the identification of more than 1700 HLA-I and 2200 HLA-II peptides. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma (IFNγ). Our analysis revealed an augmented presentation of chymotryptic-like and longer ligands associated with IFNγ induced changes of the antigen processing and presentation machinery. This straightforward method is applicable for basic and clinical applications.

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.