Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation.
Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation.
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DOI：
10.1074/mcp.m114.042812
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发表时间：
2015-03
期刊：
影响因子：
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通讯作者：
Mann M
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文献类型：
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作者：
Bassani-Sternberg M;Pletscher-Frankild S;Jensen LJ;Mann M
HLA I类分子通过呈递内源性衍生肽的库来反映细胞对细胞毒性T细胞的健康状态。然而，蛋白质组在多大程度上塑造肽组仍然是未知的。在这里，我们提出了一个高通量的质谱为基础的工作流程，允许严格和准确的识别数以千计的这样的肽和结合基序的直接测定。将该工作流程应用于七种癌细胞系和原代细胞，产生了超过22，000种不同等位基因结合特异性的独特HLA肽。通过计算代表HLA-I采样密度的分数，我们显示了蛋白质丰度和HLA呈递之间的强关联（p < 0.0001）。当分析过提呈蛋白质时-那些密度评分比预期的丰度高至少5倍的蛋白质-我们注意到它们的降解速度比类似但未提呈的蛋白质快近3小时（前20%丰度类别;中位半衰期20.8小时对23.6小时，p < 0.0001）。这证实了蛋白质降解是HLA呈递的重要因素。核糖体，线粒体呼吸链，和核小体蛋白质是特别好的介绍。以一组与癌症相关的蛋白质为例，我们将先前验证的T细胞表位的预测免疫原性与我们数据集中这些蛋白质的其他肽进行了比较。经验证的表位确实倾向于具有比其他检测到的HLA肽更高的免疫原性评分。值得注意的是，我们从人结肠癌细胞系中鉴定出了五种突变肽，最近预测它们是HLA-I结合剂。总之，我们证明了MS分析与免疫发生预测相结合的有用性，用于识别，排名和选择用于治疗用途的肽。
HLA class I molecules reflect the health state of cells to cytotoxic T cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (p < 0.0001). When analyzing overpresented proteins – those with at least fivefold higher density score than expected for their abundance – we noticed that they are degraded almost 3 h faster than similar but nonpresented proteins (top 20% abundance class; median half-life 20.8h versus 23.6h, p < 0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain, and nucleosomal proteins are particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our data set. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides. Remarkably, we identified five mutated peptides from a human colon cancer cell line, which have very recently been predicted to be HLA-I binders. Altogether, we demonstrate the usefulness of combining MS-analysis with immunogenesis prediction for identifying, ranking, and selecting peptides for therapeutic use.