Du Zhuokun, Yang Qianying, Liu Yuanyuan, Chen Sijie, Zhao Hongxian, Bai Haihong, Shao Wei, Zhang Yangjun, Qin Weijie
School of Basic Medical Science, Anhui Medical University, Hefei, China.
State Key Laboratory of Proteomics, National Center for Protein Sciences Beijing, Beijing Institute of Lifeomics, Beijing Proteome Research Center, Beijing, China.
Front Mol Biosci. 2022 May 24;9:923363. doi: 10.3389/fmolb.2022.923363. eCollection 2022.
N-glycosylation and phosphorylation, two common posttranslational modifications, play important roles in various biological processes and are extensively studied for biomarker and drug target screening. Because of their low abundance, enrichment of N-glycopeptides and phosphopeptides prior to LC-MS/MS analysis is essential. However, simultaneous characterization of these two types of posttranslational modifications in complex biological samples is still challenging, especially for tiny amount of samples obtained in tissue biopsy. Here, we introduced a new strategy for the highly efficient tandem enrichment of N-glycopeptides and phosphopeptides using HILIC and TiO microparticles. The N-glycopeptides and phosphosites obtained by tandem enrichment were 21%-377% and 22%-263% higher than those obtained by enriching the two PTM peptides separately, respectively, using 160-20 μg tryptic digested peptides as the starting material. Under the optimized conditions, 2798 N-glycopeptides from 434 N-glycoproteins and 5130 phosphosites from 1986 phosphoproteins were confidently identified from three technical replicates of HeLa cells by mass spectrometry analysis. Application of this tandem enrichment strategy in a lung cancer study led to simultaneous characterization of the two PTM peptides and discovery of hundreds of differentially expressed N-glycosylated and phosphorylated proteins between cancer and normal tissues, demonstrating the high sensitivity of this strategy for investigation of dysregulated PTMs using very limited clinical samples.
N-糖基化和磷酸化是两种常见的翻译后修饰，在各种生物学过程中发挥着重要作用，并且在生物标志物和药物靶点筛选方面得到了广泛研究。由于它们的丰度较低，在液相色谱-串联质谱（LC-MS/MS）分析之前对N-糖肽和磷酸肽进行富集至关重要。然而，在复杂生物样品中同时表征这两种类型的翻译后修饰仍然具有挑战性，特别是对于组织活检中获得的微量样品。在此，我们介绍了一种使用亲水相互作用色谱（HILIC）和二氧化钛（TiO）微粒对N-糖肽和磷酸肽进行高效串联富集的新策略。以160 - 20μg胰蛋白酶消化的肽段为起始材料，通过串联富集获得的N-糖肽和磷酸化位点分别比单独富集这两种翻译后修饰肽段时获得的高出21% - 377%和22% - 263%。在优化条件下，通过质谱分析从HeLa细胞的三个技术重复中可靠地鉴定出了来自434种N-糖蛋白的2798个N-糖肽和来自1986种磷酸化蛋白的5130个磷酸化位点。这种串联富集策略在肺癌研究中的应用导致同时表征了这两种翻译后修饰肽段，并发现了癌症组织和正常组织之间数百种差异表达的N-糖基化和磷酸化蛋白，证明了该策略在使用非常有限的临床样品研究失调的翻译后修饰方面具有高灵敏度。
