Simultaneous enrichment and sequential separation of glycopeptides and phosphopeptides with poly-histidine functionalized microspheres.
Simultaneous enrichment and sequential separation of glycopeptides and phosphopeptides with poly-histidine functionalized microspheres.
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DOI：
10.3389/fbioe.2022.1011851
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发表时间：
2022
影响因子：
5.7
通讯作者：
Liang, Xinmiao
中科院分区：
文献类型：
--
作者：
Shang, Danyi;Chen, Cheng;Dong, Xuefang;Cui, Yun;Qiao, Zichun;Li, Xiuling;Liang, Xinmiao
蛋白质的磷酸化和糖基化协调调节着许多复杂的生物过程。然而，目前主要的方法是基于配位相互作用或Lewis酸碱相互作用，但由于分子间相互作用较强，目标分子覆盖率较低。在这里，我们构建了一种基于多组氨酸修饰的二氧化硅(SiO_2@Poly-His)微球的方法，用于同时浓缩、顺序洗脱和分析磷酸肽和糖肽。由亲水相互作用和多重氢键相互作用驱动的SiO_2@Poly-His微球对1,000摩尔倍数的牛血清白蛋白干扰中的磷酸肽和糖肽具有高的选择性和覆盖率。此外，“在线脱糖”策略允许磷酸肽和糖肽的顺序洗脱，保护磷酸肽在脱糖基化过程中不被水解，并提高磷酸肽的覆盖率。将我们建立的方法应用于HT29细胞裂解产物，总共得到1,601个鉴定的糖肽和694个鉴定的磷酸肽，分别比共洗脱策略得到的高1.2倍和1.5倍。基于SiO_2@Poly-His的同时浓缩和顺序分离策略在生物和临床样品的PTMS-蛋白质组学的联合分析中具有很大的潜力。
Protein phosphorylation and glycosylation coordinately regulate numerous complex biological processes. However, the main methods to simultaneously enrich them are based on the coordination interactions or Lewis acid-base interactions, which suffer from low coverage of target molecules due to strong intermolecular interactions. Here, we constructed a poly-histidine modified silica (SiO2@Poly-His) microspheres-based method for the simultaneous enrichment, sequential elution and analysis of phosphopeptides and glycopeptides. The SiO2@Poly-His microspheres driven by hydrophilic interactions and multiple hydrogen bonding interactions exhibited high selectivity and coverage for simultaneous enrichment of phosphopeptides and glycopeptides from 1,000 molar folds of bovine serum albumin interference. Furthermore, “on-line deglycosylation” strategy allows sequential elution of phosphopeptides and glycopeptides, protecting phosphopeptides from hydrolysis during deglycosylation and improving the coverage of phosphopeptides. The application of our established method to HT29 cell lysates resulted in a total of 1,601 identified glycopeptides and 694 identified phosphopeptides, which were 1.2-fold and 1.5-fold higher than those obtained from the co-elution strategy, respectively. The SiO2@Poly-His based simultaneous enrichment and sequential separation strategy might have great potential in co-analysis of PTMs-proteomics of biological and clinic samples.
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