Anal Chem. 2020 Jul 7;92(13):9194-9204. doi: 10.1021/acs.analchem.0c01545. Epub 2020 Jun 23.
Defining the repertoire of peptides presented by the major histocompatibility complex class I (MHC I) is a key step toward the identification of relevant antigens for cancer immunotherapy. However, the identification of cancer-specific antigens is a significant analytical challenge in view of their low abundance and low mutational load found in most primary cancer specimens. Here, we describe the application of isobaric peptide labeling with tandem mass tag (TMT) to improve the detection of the MHC I peptides. Isobaric peptide labeling was found to promote the formation of multiply charged ions and to enhance the formation of b-type fragment ions, thus resulting in a 50% improvement of MHC I peptide identification. The gain in sensitivity obtained using TMT labeling enabled the detection of low-abundance MHC I peptides including tumor-specific antigens (TSAs) and minor histocompatibility antigens (MiHAs). We further demonstrate the application of this approach to quantify MiHAs presented by B-cell lymphocytes and determined their expression levels by LC-MS/MS using both synchronous precursor selection (SPS) and high-field asymmetric waveform ion mobility spectrometry (FAIMS).
定义主要组织相容性复合体 I（MHC I）呈递的肽库是鉴定癌症免疫治疗相关抗原的关键步骤。然而，鉴于大多数原发性癌症标本中抗原的低丰度和低突变负荷，鉴定癌症特异性抗原是一个重大的分析挑战。在这里，我们描述了使用同位素质谱标签（TMT）进行等压肽标记的应用，以提高 MHC I 肽的检测。等压肽标记被发现可以促进多电荷离子的形成，并增强 b 型碎片离子的形成，从而使 MHC I 肽鉴定的灵敏度提高了 50%。使用 TMT 标记获得的灵敏度提高能够检测低丰度的 MHC I 肽，包括肿瘤特异性抗原（TSA）和次要组织相容性抗原（MiHA）。我们进一步证明了该方法在定量 B 细胞淋巴细胞呈递的 MiHA 中的应用，并使用同步前体选择（SPS）和高场非对称波形离子迁移谱（FAIMS）通过 LC-MS/MS 测定了它们的表达水平。
