Chair of Proteomics and Bioanalytics, Technical University of Munich (TUM), Freising 85354, Germany.
Institute of Pathology, Technical University of Munich (TUM), Munich 81675, Germany.
J Proteome Res. 2021 Dec 3;20(12):5402-5411. doi: 10.1021/acs.jproteome.1c00695. Epub 2021 Nov 4.
Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving a high proteomic depth and limiting the overall analysis time. To this end, we evaluated the merits of online coupling of single-use disposable trap column nanoflow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS), and tandem mass spectrometry (nLC-FAIMS-MS/MS). The data show that ≤600 ng of peptide digest should be loaded onto the chromatographic part of the system. Careful characterization of the FAIMS settings enabled the choice of optimal combinations of compensation voltages (CVs) as a function of the employed LC gradient time. We found nLC-FAIMS-MS/MS to be on par with StageTip-based off-line basic pH reversed-phase fractionation in terms of proteomic depth and reproducibility of protein quantification (coefficient of variation ≤15% for 90% of all proteins) but requiring 50% less sample and substantially reducing sample handling. Using FFPE materials from the lymph node, lung, and prostate tissue as examples, we show that nLC-FAIMS-MS/MS can identify 5000-6000 proteins from the respective tissue within a total of 3 h of analysis time.
使用福尔马林固定石蜡包埋（FFPE）组织进行蛋白质组学生物标志物发现需要强大的工作流程来支持对大量患者样本进行分析。它还需要在实现高蛋白质组深度和限制整体分析时间之间找到合理的平衡。为此，我们评估了在线连接一次性使用的捕获柱纳流液相色谱、高场非对称波形离子迁移谱（FAIMS）和串联质谱（nLC-FAIMS-MS/MS）的优点。数据表明，应将≤600ng 的肽消化物加载到系统的色谱部分。仔细表征 FAIMS 设置可选择最佳补偿电压（CV）组合，作为所采用的 LC 梯度时间的函数。我们发现，nLC-FAIMS-MS/MS 在蛋白质组深度和蛋白质定量重现性方面与基于 StageTip 的离线碱性 pH 反相分级分离相当（所有蛋白质的 90%的 CV 值≤15%），但需要的样品减少 50%，并且大大减少了样品处理。使用淋巴结、肺和前列腺组织的 FFPE 材料作为示例，我们表明，nLC-FAIMS-MS/MS 可以在总共 3 小时的分析时间内从各自的组织中鉴定出 5000-6000 种蛋白质。
