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首页> 外文期刊>Analytical chemistry >Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis
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Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis

机译：离心辅助微反应器可实现胰蛋白酶消化，亲水相互作用色谱富集和柱上去糖基化的便捷整合，从而实现快速灵敏的N-糖蛋白组学分析

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摘要

Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80percent, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 (mu)L of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.
机译：样品处理程序包括蛋白质消化，糖肽富集和去糖基化对糖蛋白分析的性能有重要影响。开发了几种糖蛋白组学分析系统来整合其中一些样品制备程序。但是，没有微系统将上述三个过程全部集成在一起。在这项工作中，我们开发了一种糖蛋白组学微反应器，能够无缝整合所有这些程序。在该反应器中，通过添加乙腈至80％来加速胰蛋白酶消化，并且在用三氟乙酸（TFA）酸化蛋白质消化物后，依次进行以下亲水相互作用色谱（HILIC）富集和去糖基化反应，而无需进行任何脱盐，冻干或缓冲液交换脚步。总处理时间可能短至1.5小时。还获得了低至30 fmol的人IgG检测限。当应用于人血清糖蛋白组分析时，分别从10 nL，100 nL和1μL人血清的三个重复分析中鉴定出总数为92、178和221个独特的N-糖基化位点。结果表明，基于糖蛋白组微反应器的方法具有很高的灵敏度，非常适用于微小蛋白质样品的糖蛋白组分析。

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