Institute of Immunology, University Medical Center of the Johannes-Gutenberg University, Mainz, Germany.
Helmholtz Institute for Translational Oncology Mainz (HI-TRON Mainz) - A Helmholtz Institute of the DKFZ, Mainz, Germany.
Nat Commun. 2024 Mar 13;15(1):2288. doi: 10.1038/s41467-024-46380-y.
Human leukocyte antigen (HLA) class I peptide ligands (HLAIps) are key targets for developing vaccines and immunotherapies against infectious pathogens or cancer cells. Identifying HLAIps is challenging due to their high diversity, low abundance, and patient individuality. Here, we develop a highly sensitive method for identifying HLAIps using liquid chromatography-ion mobility-tandem mass spectrometry (LC-IMS-MS/MS). In addition, we train a timsTOF-specific peak intensity MSPIP model for tryptic and non-tryptic peptides and implement it in MSRescore (v3) together with the CCS predictor from ionmob. The optimized method, Thunder-DDA-PASEF, semi-selectively fragments singly and multiply charged HLAIps based on their IMS and m/z. Moreover, the method employs the high sensitivity mode and extended IMS resolution with fewer MS/MS frames (300 ms TIMS ramp, 3 MS/MS frames), doubling the coverage of immunopeptidomics analyses, compared to the proteomics-tailored DDA-PASEF (100 ms TIMS ramp, 10 MS/MS frames). Additionally, rescoring boosts the HLAIps identification by 41.7% to 33%, resulting in 5738 HLAIps from as little as one million JY cell equivalents, and 14,516 HLAIps from 20 million. This enables in-depth profiling of HLAIps from diverse human cell lines and human plasma. Finally, profiling JY and Raji cells transfected to express the SARS-CoV-2 spike protein results in 16 spike HLAIps, thirteen of which have been reported to elicit immune responses in human patients.
人类白细胞抗原 (HLA) I 类肽配体 (HLAIps) 是针对传染病原体或癌细胞开发疫苗和免疫疗法的关键靶标。由于其多样性高、丰度低和个体差异大，因此鉴定 HLAIps 具有挑战性。在这里，我们使用液相色谱-离子淌度-串联质谱 (LC-IMS-MS/MS) 开发了一种高度敏感的 HLAIps 鉴定方法。此外，我们还针对胰蛋白酶和非胰蛋白酶肽训练了一个 timsTOF 特定的峰强度 MSPIP 模型，并将其与来自 ionmob 的 CCS 预测器一起在 MSRescore (v3) 中实现。优化后的方法 Thunder-DDA-PASEF 根据其 IMS 和 m/z 对单电荷和多电荷 HLAIps 进行半选择性片段化。此外，该方法采用高灵敏度模式和扩展的 IMS 分辨率，使用较少的 MS/MS 帧数 (300 ms TIMS 斜坡，3 个 MS/MS 帧数)，与针对蛋白质组学的 DDA-PASEF(100 ms TIMS 斜坡，10 个 MS/MS 帧数)相比，免疫肽组学分析的覆盖率增加了一倍。此外，重新评分将 HLAIps 的鉴定率提高了 41.7%至 33%，从仅 100 万个 JY 细胞当量中鉴定出 5738 个 HLAIps，从 2000 万个细胞中鉴定出 14516 个 HLAIps。这使得能够深入分析来自不同人类细胞系和人类血浆的 HLAIps。最后，对表达 SARS-CoV-2 刺突蛋白的 JY 和 Raji 细胞进行分析，结果鉴定出 16 个刺突 HLAIps，其中 13 个已被报道在人类患者中引起免疫反应。
