Mass Spectrometry for Biology Unit, CNRS USR2000, Institut Pasteur, Paris 75015, France.
Microbiology Department, Georges Pompidou European Hospital, Assistance Publique-Hôpitaux de Paris, Paris 75015, France.
J Proteome Res. 2021 Jan 1;20(1):202-211. doi: 10.1021/acs.jproteome.0c00351. Epub 2020 Oct 1.
The current technique used for microbial identification in hospitals is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, it suffers from important limitations, in particular, for closely related species or when the database used for the identification lacks the appropriate reference. In this work, we set up a liquid chromatography (LC)-MS/MS top-down proteomics platform, which aims at discriminating closely related pathogenic bacteria through the identification of specific proteoforms. Using as a model, all steps of the workflow were optimized: protein extraction, on-line LC separation, MS method, and data analysis. Using optimized parameters, about 220 proteins, corresponding to more than 500 proteoforms, could be identified in a single run. We then used this platform for the discrimination of enterobacterial pathogens undistinguishable by MALDI-TOF, although leading to very different clinical outcomes. For each pathogen, we identified specific proteoforms that could potentially be used as biomarkers. We also improved the characterization of poorly described bacterial strains. Our results highlight the advantage of addressing proteoforms rather than peptides for accurate bacterial characterization and qualify top-down proteomics as a promising tool in clinical microbiology. Data are available ProteomeXchange with the identifier PXD019247.
目前医院中用于微生物鉴定的技术是基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）。然而，它存在重要的局限性，特别是对于密切相关的物种，或者当用于鉴定的数据库缺乏适当的参考时。在这项工作中，我们建立了一个液相色谱（LC）-MS/MS 自上而下的蛋白质组学平台，旨在通过鉴定特定的蛋白质形式来区分密切相关的致病菌。使用 作为模型，我们对工作流程的所有步骤进行了优化：蛋白质提取、在线 LC 分离、MS 方法和数据分析。使用优化的参数，在单个运行中可以鉴定出约 220 种蛋白质，对应于超过 500 种蛋白质形式。然后，我们使用该平台来区分 MALDI-TOF 无法区分的肠杆菌病原体，尽管它们导致非常不同的临床结果。对于每种病原体，我们都鉴定了可能用作生物标志物的特定蛋白质形式。我们还改进了对描述不佳的细菌菌株的特征描述。我们的结果强调了针对蛋白质形式而不是肽进行准确细菌特征描述的优势，并将自上而下的蛋白质组学确定为临床微生物学中很有前途的工具。数据可在 ProteomeXchange 中以标识符 PXD019247 获得。
