Optimization of Microflow LC Coupled with Scanning SWATH and Its Application in Hepatocellular Carcinoma Tissues
Optimization of Microflow LC Coupled with Scanning SWATH and Its Application in Hepatocellular Carcinoma Tissues
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微流LC联用扫描SWATH的优化及其在肝细胞癌组织中的应用
DOI：
10.1021/acs.jproteome.2c00078
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发表时间：
2022
影响因子：
4.4
通讯作者：
Yi Zhu
中科院分区：
文献类型：
--
作者：
Huanhuan Gao;Youqi Liu;Vadim Demichev;Stephen Tate;Chen Chen;Jiang Zhu;Cong Lu;Markus Ralser;Tiannan Guo;Yi Zhu
扫描SWATH结合正常流动LC最近已被引入用于高含量、高通量蛋白质组学分析，这需要相对大量的样品注射。在这里，我们建立了与扫描SWATH耦合的微流LC，用于相对少量的样品。首先，我们优化了LC和MS设置的几个关键参数，包括分析柱的C18粒度、LC梯度和流速，以及MS采集的有效离子积累时间和隔离窗口宽度。然后，我们比较了优化的扫描SWATH方法与传统的可变窗口SWATH（简称SWATH）方法。结果表明，扫描SWATH中的总离子色谱信号比SWATH高10倍，扫描SWATH比SWATH多识别12.2-22.2%的肽。最后，我们采用120分钟扫描SWATH获得的蛋白质组的62福尔马林固定，石蜡包埋（FFPE）组织样本从31例肝细胞癌（HCC）。总共定量了92334种肽和8516种蛋白质。除了已报道的生物标志物，包括ANXA 2、MCM 7、SUOX和AKR 1B 10，我们还鉴定了新的潜在HCC生物标志物，如CST 5、TP 53、CEBPB和E2 F4。综上所述，我们提出了一种集成微流LC和扫描SWATH的最佳工作流程，有效地提高了蛋白质的鉴定和定量。
Scanning SWATH coupled with normal-flow LC has been recently introduced for high-content, high-throughput proteomics analysis, which requires a relatively large amount of sample injection. Here we established the microflow LC coupled with Scanning SWATH for samples with relatively small quantities. First, we optimized several key parameters of the LC and MS settings, including C18 particle size for the analytical column, LC gradient and flow rate, as well as effective ion accumulation time and isolation window width for MS acquisition. We then compared the optimized Scanning SWATH method with the conventional variable window SWATH (referred to as SWATH) method. Results showed that the total ion chromatogram signals in Scanning SWATH were 10 times higher than that of SWATH, and Scanning SWATH identified 12.2-22.2% more peptides than SWATH. Finally, we employed 120 min Scanning SWATH to acquire the proteomes of 62 formalin-fixed, paraffin-embedded (FFPE) tissue samples from 31 patients with hepatocellular carcinoma (HCC). Altogether, 92 334 peptides and 8516 proteins were quantified. Besides the reported biomarkers, including ANXA2, MCM7, SUOX, and AKR1B10, we identified new potential HCC biomarkers such as CST5, TP53, CEBPB, and E2F4. Taken together, we present an optimal workflow integrating microflow LC and Scanning SWATH that effectively improves the protein identification and quantitation.