Spectronaut and DIA-NN: A Comparison of their Performance in the Analysis of Lung Adenocarcinoma Biopsies

Direct data-independent acquisition (DIA) in liquid chromatography–tandem mass spectrometry (LC–MS/MS) has developed into a powerful methodology for proteomics. Herein, we directly compare the performance of two widely used DIA computational platformsSpectronaut and DIA-NNusing paired tumor and peritumor tissue biopsies from human lung carcinoma (LUAD) patients, samples that one would encounter in a clinical research lab in a hospital setting. The evaluations include the following: (1) protein identification depth, (2) quantitative consistency, and (3) differential expression on the same set of LC–MS/MS runs, using nominally identical computational parameters and settings. Both Spectronaut and DIA-NN identified ∼>7600 proteins in the LUAD samples, with 7180 proteins common to both platforms. Spectronaut reported 1250 upregulated proteins and 266 downregulated proteins (tumor versus peritumor), while DIA-NN reported 1819 and 174 proteins, respectively. A total of 1130 differentially expressed proteins (DEPs) were common to both platforms. Of the top 50 DEPs, 34 were shared between Spectronaut and DIA-NN. Two of these DEPsHSPA5 and CAV1were also among the top 50 proteins showing the highest degrees of protein–protein interaction. The DEPs enabled classification of the LUAD patients into three subtypes; the clinical validity of the current subtypes will need to be substantiated in the future with additional LUAD samples and experimentation.液相色谱-串联质谱（liquid chromatography–tandem mass spectrometry, LC–MS/MS）中的直接数据非依赖采集（data-independent acquisition, DIA）现已发展成为蛋白质组学领域极具潜力的研究方法。本研究采用人类肺腺癌（lung adenocarcinoma, LUAD）患者的配对肿瘤与癌旁组织活检样本——即医院临床研究实验室中常见的样本类型——直接对比两款主流DIA分析计算平台——Spectronaut与DIA-NN——的性能表现。本次评估基于同一批LC–MS/MS上机数据，采用标称一致的计算参数与配置，涵盖以下三个维度：(1) 蛋白质鉴定深度；(2) 定量一致性；(3) 差异表达分析。两款平台在LUAD样本中均鉴定出约7600余种蛋白质，其中共有蛋白质达7180个。针对肿瘤组与癌旁组的对比分析，Spectronaut鉴定得到1250个上调蛋白与266个下调蛋白，而DIA-NN则分别得到1819个上调蛋白与174个下调蛋白。两款平台共鉴定得到1130个共有差异表达蛋白（differentially expressed proteins, DEPs）。在排名前50的DEPs中，两款平台共有34个。其中HSPA5与CAV1这两个DEPs，同时跻身蛋白质相互作用强度排名前50的蛋白质行列。上述DEPs可将LUAD患者划分为三个亚型，但当前亚型的临床有效性仍需未来通过更多LUAD样本与实验研究加以验证。