基于质谱的免疫肽组学是唯一无偏见的方法，能够鉴定自然显示的HLA配体，这是表征新型肿瘤抗原以进行免疫治疗的必要前提。近年来，基于设备和方法的改进已经增加了免疫肽组学的灵敏度和通量。然而，配体分离、质谱分析和后续数据处理方面的发展可能会对免疫肽组学数据的质量和数量产生显著影响。本研究中，我们使用细胞系以及原发性实体肿瘤和血液肿瘤样本，比较了两种已建立的免疫沉淀方法（基于柱和基于96孔板）在肽产量、光谱质量、疏水性、滞留时间和免疫原性方面的组成。尽管我们发现了相当的肽产量，但柱法和专有方法之间存在较大比例的独占肽，其疏水性显著较高，这可能对免疫原性肿瘤抗原的鉴定产生潜在影响。我们表明，柱制备在亲水洗涤步骤中不会损失亲水性肽。相反，在96孔板制备过程中，额外的50%乙腈洗脱可部分恢复失去的疏水性肽，这表明减少了对柱法的偏倚，但并未完全均衡。总之，本研究展示了不同的免疫沉淀方法及其改进如何影响免疫肽组分析的肽库，从而影响潜在肿瘤相关抗原的鉴定。版权所有 © 2023 Wacker, Bauer, Wessling, Dubbelaar, Nelde, Rammensee和Walz。

Mass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for characterizing novel tumor antigens for immunotherapeutic approaches. In recent years, improvements based on devices and methodology have been made to optimize sensitivity and throughput in immunopeptidomics. However, developments in ligand isolation, mass spectrometric analysis, and subsequent data processing can have a marked impact on the quality and quantity of immunopeptidomics data.In this work, we compared the immunopeptidome composition in terms of peptide yields, spectra quality, hydrophobicity, retention time, and immunogenicity of two established immunoprecipitation methods (column-based and 96-well-based) using cell lines as well as primary solid and hematological tumor samples.Although, we identified comparable overall peptide yields, large proportions of method-exclusive peptides were detected with significantly higher hydrophobicity for the column-based method with potential implications for the identification of immunogenic tumor antigens. We showed that column preparation does not lose hydrophilic peptides in the hydrophilic washing step. In contrast, an additional 50% acetonitrile elution could partially regain lost hydrophobic peptides during 96-well preparation, suggesting a reduction of the bias towards the column-based method but not completely equalizing it.Together, this work showed how different immunoprecipitation methods and their adaptions can impact the peptide repertoire of immunopeptidomic analysis and therefore the identification of potential tumor-associated antigens.Copyright © 2023 Wacker, Bauer, Wessling, Dubbelaar, Nelde, Rammensee and Walz.