Pritchard Antonia L, Hastie Marcus L, Neller Michelle, Gorman Jeffrey J, Schmidt Chris W, Hayward Nicholas K
Oncogenomics Research Group, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Qld, Australia.
Pigment Cell Melanoma Res. 2015 May;28(3):281-94. doi: 10.1111/pcmr.12357. Epub 2015 Mar 5.
Advancements in high-resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13,829 peptides were identified; 83-87% of these were 8-11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA-type binding prediction for 10,078 9/10 mer peptides assigned 88-95% to a patient-specific HLA subtype, revealing a disparity in strength of predicted binding. HLA-B*27-specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.
高分辨率高效液相色谱法（HPLC）和质谱技术的进步，使得这项技术在鉴定从免疫纯化的I类主要组织相容性复合体（MHC）分子上洗脱下来的肽段方面重获活力。使用w6/32分离、肽段洗脱和HPLC纯化方法对三种黑色素瘤细胞系进行了评估；通过质谱鉴定肽段。总共鉴定出13829个肽段；其中83 - 87%为8 - 11聚体。之前仅描述过约15%。来源蛋白的亚细胞定位显示取样均匀；mRNA表达和总蛋白长度可预测从单个蛋白中检测到的肽段数量。对10078个9/10聚体肽段进行的HLA类型结合预测显示，88 - 95%归属于患者特异性HLA亚型，这揭示了预测结合强度的差异。HLA - B*27特异性分离成功鉴定出一些使用w6/32未发现的肽段。基于来源蛋白和预测的HLA结合情况，选择了60个肽段进行免疫筛选；未发现抗黑色素瘤T细胞识别的新肽段。此外，质谱无法鉴定出一名患者的T细胞在体外靶向的几个表位。
