We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.DOI: http://dx.doi.org/10.7554/eLife.07661.001
The aim of this study was to develop a novel method to detect circulating histones H3 and H2B in plasma based on multiple reaction monitoring targeted mass spectrometry and a multiple reaction monitoring approach (MRM-MS) for its clinical application in critical bacteriaemic septic shock patients. Plasma samples from 17 septic shock patients with confirmed bacteraemia and 10 healthy controls were analysed by an MRM-MS method, which specifically detects presence of histones H3 and H2B. By an internal standard, it was possible to quantify the concentration of circulating histones in plasma, which were significantly higher in patients, and thus confirmed their potential as biomarkers for diagnosing septic shock. After comparing surviving patients and non-survivors, a correlation was found between higher levels of circulating histones and unfavourable outcome. Indeed, histone H3 proved a more efficient and sensitive biomarker for septic shock prognosis. In conclusion, these findings suggest the accuracy of the MRM-MS technique and stable isotope labelled peptides to detect and quantify circulating plasma histones H2B and H3. This method may be used for early septic shock diagnoses and for the prognosis of fatal outcomes.
Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with highresolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO 2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.034314, 462-474, 2014. Major histocompatibility complex (MHC)1 class I molecules are cell surface glycoproteins that are expressed on almost every nucleated cell in vertebrates. They result from the noncovalent interaction of a polymorphic heavy chain, a constant light chain (-2-microglobulin (2m)), and a peptide ligand (1). The extracellular region of the heavy chain encompasses three domains, ␣ 1 , ␣ 2 , and ␣ 3 , with ␣ 1 and ␣ 2 forming a groove that accommodates a peptide ligand of, typically, 8 to 11 amino acid residues. The binding of the ligand to the groove is governed by the interaction of the side chains of certain peptide residues, called anchor positions, with several pockets of the heavy chain named A to F (1, 2). The size and chemical nature of these pockets impose restrictions on the peptide repertoire that can be associated with a particular class I antigen. It is reckoned that the ligandome of a given class I allotype may comprise up to 10,000 different peptides (3), although recent reports suggest that this number may be underestimated (4).Peptides displayed by MHC class I molecules derive from the intracellular degradation of endogenous proteins in the nucleus and cytosol and reach the lumen of the endoplasmic reticulum by means of the transporter associated with antigen processing. Inside the endoplasmic reticulum, peptides bind to the heavy chain and 2m in a multistep process involving several c...
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