Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.
Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.
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DOI：
10.1016/j.mcpro.2021.100133
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Carr SA
中科院分区：
文献类型：
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作者：
Klaeger S;Apffel A;Clauser KR;Sarkizova S;Oliveira G;Rachimi S;Le PM;Tarren A;Chea V;Abelin JG;Braun DA;Ott PA;Keshishian H;Hacohen N;Keskin DB;Wu CJ;Carr SA
质谱法是直接鉴定人类白细胞抗原(人类白细胞抗原)分子上的多肽最有效的方法。然而，目前的标准方法通常使用5亿或更多的细胞作为输入，以实现免疫球蛋白的高覆盖率，因此，这些方法与临床肿瘤样本中可用组织的数量往往有限不兼容。在这里，我们评估了微量基础反相分离离线分离人类白细胞抗原多肽样品，然后离子迁移率与LC-MS/MS联用进行分析。这两种分离方法的结合使得与没有预先分离或单独使用离子迁移率的分析样品相比，能够多鉴定20%到50%的多肽。我们展示了高达8107个不同的多肽的人类白细胞抗原免疫多肽的覆盖范围，这些多肽最初只有1亿个细胞。使用我们的方法获得的更高的灵敏度可以为改进人类白细胞抗原结合预测算法提供有用的数据，以及能够检测临床上相关的表位，如新抗原。使用液相和气相分离的深层免疫肽覆盖。使用微尺度基础反相分离，高达50%以上的人类白细胞抗原-I多肽。离子迁移率分离(FAIMS)提高了高达58%的人类白细胞抗原-I多肽识别能力。这些方法提供的更高的灵敏度使新抗原的检测成为可能。在这里，我们评估了离线微尺度基础反相分离以及使用离子迁移率与LC-MS/MS联用来分析在人类白细胞抗原-I上呈现的多肽。与分析样品相比，这两种分离方法可以多鉴定20%到50%的多肽，而不需要事先进行分级或仅使用离子迁移率，从最少1亿个细胞开始。使用我们的方法获得的更高的灵敏度可以检测到低丰度但临床相关的表位，如新抗原。
MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline 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 with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens. Deep immunopeptidome coverage using liquid and gas phase separation. Up to 50% more HLA-I peptides using microscaled basic reversed-phase fractionation. Ion mobility separation (FAIMS) increases HLA-I peptide identifications by up to 58%. Increased sensitivity provided by these methods enables detection of neoantigens. Here, we evaluated off-line microscaled basic reversed-phase fractionation as well as the use of ion mobility coupled to LC–MS/MS for analysis of peptides presented on HLA-I. The two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone starting with as few as 100 million cells. The increased sensitivity obtained using our methods can enable detection of low abundant but clinically relevant epitopes such as neoantigens.