Monolithic Capillary Column Based Glycoproteomic Reactor for High-Sensitive Analysis of N-Glycoproteome

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• 作者：Jing Liu ; Fangjun Wang ; Hui Lin ; Jun Zhu ; Yangyang Bian ; Kai Cheng ; Hanfa Zou
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：March 5, 2013
• 年：2013
• 卷：85
• 期：5
• 页码：2847-2852
• 全文大小：252K
• 年卷期：v.85,no.5(March 5, 2013)
• ISSN：1520-6882

文摘

Despite the importance of protein N-glycosylation in a series of biological processes, in-depth characterization of protein glycosylation is still a challenge due to the high complexity of biological samples and the lacking of highly sensitive detection technologies. We developed a monolithic capillary column based glycoproteomic reactor enabling high-sensitive mapping of N-glycosylation sites from minute amounts of sample. Unlike the conventional proteomic reactors with only strong-cation exchange or hydrophilic-interaction chromatography columns, this novel glycoproteomic reactor was composed of an 8 cm long C12 hydrophobic monolithic capillary column for protein digestion and a 6 cm long organic鈥搒ilica hybrid hydrophilic monolithic capillary column for glycopeptides enrichment and deglycosylation, which could complete whole-sample preparation including protein purification/desalting, tryptic digestion, enrichment, and deglycosylation of glycopeptides within about 3 h. The developed reactor exhibited high detection sensitivity in mapping of N-glycosylation sites by detection limit of horseradish peroxidase as low as 2.5 fmol. This reactor also demonstrated the ability in complex sample analysis, and in total, 486 unique N-glycosylation sites were reliably mapped in three replicate analyses of a protein sample extracted from 10⁴ HeLa cells.