Department of Biochemistry and Molecular Biology, Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
The Jenner Institute, Mass Spectrometry Laboratory, Target Discovery Institute, University of Oxford, Oxford, UK.
Nat Protoc. 2019 Jun;14(6):1687-1707. doi: 10.1038/s41596-019-0133-y. Epub 2019 May 15.
Peptide antigens bound to molecules encoded by the major histocompatibility complex (MHC) and presented on the cell surface form the targets of T lymphocytes. This critical arm of the adaptive immune system facilitates the eradication of pathogen-infected and cancerous cells, as well as the production of antibodies. Methods to identify these peptide antigens are critical to the development of new vaccines, for which the goal is the generation of effective adaptive immune responses and long-lasting immune memory. Here, we describe a robust protocol for the identification of MHC-bound peptides from cell lines and tissues, using nano-ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (nUPLC-MS/MS) and recent improvements in methods for isolation and characterization of these peptides. The protocol starts with the immunoaffinity capture of naturally processed MHC-peptide complexes. The peptides dissociate from the class I human leukocyte antigens (HLAs) upon acid denaturation. This peptide cargo is then extracted and separated into fractions by HPLC, and the peptides in these fractions are identified using nUPLC-MS/MS. With this protocol, several thousand peptides can be identified from a wide variety of cell types, including cancerous and infected cells and those from tissues, with a turnaround time of 2-3 d.
与主要组织相容性复合体 (MHC) 编码的分子结合并呈现在细胞表面的肽抗原构成了 T 淋巴细胞的靶标。适应性免疫系统的这一关键部分有助于清除病原体感染和癌细胞，以及产生抗体。鉴定这些肽抗原的方法对于新疫苗的开发至关重要，其目标是产生有效的适应性免疫反应和持久的免疫记忆。在这里，我们描述了一种从细胞系和组织中鉴定 MHC 结合肽的强大方案，该方案结合使用纳升超高效液相色谱与高分辨率质谱 (nUPLC-MS/MS) 以及这些肽的分离和鉴定方法的最新改进。该方案首先从天然加工的 MHC-肽复合物中进行免疫亲和捕获。肽在酸变性时从人类白细胞抗原 (HLA) Ⅰ类解离。然后通过 HPLC 将这些肽从 HLA 中提取并分离成馏分，并使用 nUPLC-MS/MS 鉴定这些馏分中的肽。使用该方案，可以从包括癌细胞和感染细胞以及组织细胞在内的各种细胞类型中鉴定出数千种肽，周转时间为 2-3 天。
