Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA.
Pharmacology Department and.
JCI Insight. 2020 Oct 2;5(19):141264. doi: 10.1172/jci.insight.141264.
Identification of MHC class I-bound peptides by immunopurification of MHC complexes and subsequent analysis by mass spectrometry is crucial for understanding T cell immunology and immunotherapy. Investigation of the steps for the MHC ligand isolation process revealed biases in widely used isolation techniques toward peptides of lower hydrophobicity. As MHC ligand hydrophobicity correlates positively with immunogenicity, identification of more hydrophobic MHC ligands could potentially lead to more effective isolation of immunogenic peptides as targets for immunotherapies. We solved this problem by use of higher concentrations of acetonitrile for the separation of MHC ligands and their respective complexes. This increased overall MHC ligand identifications by 2-fold, increased detection of cancer germline antigen-derived peptides by 50%, and resulted in profound variations in isolation efficacy between different MHC alleles correlating with the hydrophobicity of their anchor residues. Overall, these insights enabled a more complete view of the immunopeptidome and overcame a systematic underrepresentation of these critical MHC ligands of high hydrophobicity.
通过免疫沉淀 MHC 复合物并随后进行质谱分析来鉴定 MHC 类 I 结合肽对于理解 T 细胞免疫学和免疫疗法至关重要。对 MHC 配体分离过程的步骤的研究表明，广泛使用的分离技术对疏水性较低的肽存在偏见。由于 MHC 配体的疏水性与免疫原性呈正相关，因此鉴定出更疏水的 MHC 配体可能会导致更有效地分离免疫原性肽作为免疫疗法的靶标。我们通过使用更高浓度的乙腈来分离 MHC 配体及其各自的复合物来解决这个问题。这使 MHC 配体的总体鉴定增加了 2 倍，增加了对癌症种系抗原衍生肽的检测 50%，并且导致不同 MHC 等位基因之间的分离效果存在明显差异，这与它们的锚定残基的疏水性相关。总的来说，这些见解使我们能够更全面地了解免疫肽组，并克服了对这些具有高疏水性的关键 MHC 配体的系统代表性不足。
