To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC class I peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC) and less often by mild acid elution (MAE). MAE may provide a cheap alternative to MHC-IAC for suspension cells but has been hampered by the high number of contaminating, MHC-unrelated peptides. Here, we optimized MAE, yielding MHC peptide ligand purities of more than 80%. When compared with MHC-IAC, obtained peptides were similar in numbers, identities, and to a large extent intensities, while the percentage of cysteinylated peptides was 5 times higher in MAE. The latter benefitted the discovery of MHC-allotype-specific, distinct cysteinylation frequencies at individual positions of MHC peptide ligands. MAE revealed many MHC ligands with unmodified, N-terminal cysteine residues which get lost in MHC-IAC workflows. The results support the idea that MAE might be particularly valuable for the high-confidence analysis of post-translational modifications by avoiding the exposure of the investigated peptides to enzymes and reactive molecules in the cell lysate. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells.

中文翻译：

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温和的酸洗脱和MHC免疫亲和层析显示出相似的特征，尽管HLA I类免疫肽组的特征不完全相同

为了理解和治疗免疫学相关疾病，主要组织相容性复合物（MHC）肽配体的全面，无偏性表征至关重要。在通过质谱分析之前，通常通过MHC免疫亲和色谱（MHC-IAC）分离MHC I类肽配体，而通过弱酸洗脱（MAE）分离的可能性较小。对于悬浮细胞，MAE可能是MHC-IAC的廉价替代品，但由于大量污染性MHC不相关肽而受到阻碍。在这里，我们优化了MAE，产生的MHC肽配体纯度超过80％。与MHC-IAC相比，获得的肽在数量，特性和强度上都相似，而MAE中的半胱氨酸化肽的百分比高5倍。后者有益于发现MHC特定型的人，在MHC肽配体的各个位置有不同的半胱氨酸化频率。MAE揭示了许多带有未修饰的N端半胱氨酸残基的MHC配体，这些配体在MHC-IAC工作流程中丢失了。结果支持这样的想法，即MAE通过避免所研究的肽暴露于细胞裂解液中的酶和反应性分子而对翻译后修饰的高可信度分析特别有价值。我们经过改进且经过精心记录的MAE工作流程代表了MHC-IAC用于悬浮细胞的高质量，高性价比的替代产品。结果支持这样的想法，即MAE通过避免所研究的肽暴露于细胞裂解液中的酶和反应性分子而对翻译后修饰的高可信度分析特别有价值。我们经过改进且经过精心记录的MAE工作流程代表了MHC-IAC用于悬浮细胞的高质量，高性价比的替代产品。结果支持这样的想法，即MAE通过避免所研究的肽暴露于细胞裂解液中的酶和反应性分子而对翻译后修饰的高可信度分析特别有价值。我们经过改进且经过精心记录的MAE工作流程代表了MHC-IAC用于悬浮细胞的高质量，高性价比的替代产品。