Optimized liquid and gas phase fractionations increase HLA-peptidome coverage for primary cell and tissue samples

Mass spectrometry is the most effective method to directly identify peptides presented on HLA molecules. However, current standard approaches often require billions of cells for input material to achieve high coverage of the immunopeptidome and are therefore not compatible with the often limiting amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples off-line followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared to samples analyzed without either prior fractionation or use of ion mobility. We demonstrate coverage of HLA immunopeptidomes with up to 8,107 distinct peptides starting with as few as 50 million cells or 150 milligrams of wet weight tumor tissue. This increased sensitivity can improve HLA binding prediction algorithms and enable detection of clinically relevant epitopes such as neoantigens

质谱法是直接鉴定人类白细胞抗原（HLA）呈递肽段的最有效手段。然而，现行标准方法通常需以数十亿个细胞作为起始材料，方能实现免疫肽组（immunopeptidome）的高覆盖度，因此无法适配临床肿瘤样本中普遍有限的组织体量。本研究针对人类白细胞抗原肽段样本，评估了先采用微尺度碱性反相分级分离技术进行离线分离，再结合离子淌度与液相色谱-串联质谱（LC-MS/MS）开展分析的实验方案。相较于未开展预先分级分离或未使用离子淌度的分析样本，这两种分离手段的联用可使鉴定得到的肽段数量提升20%至50%。我们证实，仅以5000万个细胞或150毫克肿瘤湿重组织作为起始材料，即可实现覆盖最多8107种独特肽段的HLA免疫肽组分析。这种灵敏度的提升能够优化HLA结合预测算法，并助力检测新抗原（neoantigens）等临床相关表位。