Integrated Sample Pretreatment System for N-Linked Glycosylation Site Profiling with Combination of Hydrophilic Interaction Chromatography and PNGase F Immobilized Enzymatic Reactor via a Strong Catio

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• 作者：Yanyan Qu ; Simin Xia ; Huiming Yuan ; Qi Wu ; Man Li ; Lijuan Zou ; Lihua Zhang ; Zhen Liang ; Yukui Zhang
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：October 1, 2011
• 年：2011
• 卷：83
• 期：19
• 页码：7457-7463
• 全文大小：897K
• 年卷期：v.83,no.19(October 1, 2011)
• ISSN：1520-6882

文摘

An integrated sample pretreatment system, composed of a click maltose hydrophilic interaction chromatography (HILIC) column, a strong cation exchange (SCX) precolumn, and a PNGase F immobilized enzymatic reactor (IMER), was established for the simultaneous glycopeptide enrichment, sample buffer exchange, and online deglycosylation, by which the sample pretreatment for glycoproteome could be performed online automatically, beneficial to improve the efficiency and sensitivity of the N-linked glycosylation site identification. With such a system, the deglycosylated glycopeptide from the digests of avidin with the coexistence of 50 times (mass ratio) BSA could be selectively detected, and the detection limit as low as 5 fmol was achieved. Moreover, the sample pretreatment time was significantly shortened to 1 h. Such a system was further successfully applied for analyzing the digest of the soluble fraction extracted from rat brain. A total of 120 unique glycoprotein groups and 196 N-linked glycosylation sites were identified by nanoreversed phase liquid chromatography鈥揺lectrospray ionization-tandem mass spectrometry (nanoRPLC-ESI-MS/MS), with the injected digests amount as 6 渭g. All these results demonstrate that the integrated system is of great promise for N-linked glycosylation site profiling and could be further online coupled with nanoHPLC-ESI-MS/MS to achieve high-throughput glycoproteome analysis.