Because of the low abundance of glycoprotein and glycopeptide in complex biological samples,it is urgent to develop an efficient method for glycopeptide enrichment in comprehensive and in-depth glycoproteomes research.Herein,a novel hydrophilic silica was developed through surface modification with cysteine-click maltose (Cys-Mal@SiO2).The developed hydrophilic silica was packed into a solid phase extraction (SPE) column,and applied to the highly selective enrichment and identification of N-linked glycopeptides.The Cys-Mal@SiO2 demonstrated better identification capability over Cys@SiO2,Mal@SiO2 and commercial hydrophilic interaction liquid chromatography (HILIC) in glycopeptide enrichment due to the synergistic effect of the two kinds of hydrophilic molecules.In the selective enrichment of tryptic digest from human immunoglobulin G,glycopeptides with higher signal-to-noises were detected by Cys-Mal@SiO2.In addition,1551 unique glycopeptides with 906 N-glycosylation sites from 466 different N-linked glycoproteins were identified from the proteins extracted from mouse liver after the enrichment with Cys-Mal@SiO2.In contrast,the numbers of identified glycopeptides,glycoproteins and N-glycosylation sites identified by Cys@SiO2 were 211,67,127 respectively less than by Cys-Mal@SiO2,and the corresponding numbers were 289,76,193 by Mal@SiO2.These results showed that the developed Cys-Mal@SiO2 is a promising affinity material for N-glycoproteomics research of real complex biological samples.%糖蛋白与糖肽在复杂生物体系中丰度一般较低,为了在糖蛋白质组学研究中深入和全面分析鉴定糖基化位点与糖链,通常需要进行富集前处理操作.该文设计并合成了一种半胱氨酸基麦芽糖修饰亲水硅胶分离介质(Cys-Mal@SiO2),将其装填入固相萃取柱中制备新型亲水固相萃取柱.在以人免疫球蛋白G酶解液为样品进行富集鉴定时,Cys-Mal@SiO2鉴定糖肽的质谱信号强度和信噪比比半胱氨酸修饰硅胶(Cys@SiO2)、麦芽糖修饰硅胶(Mal@SiO2)和商品化ZIC(R)-HILIC更高.在复杂的鼠肝蛋白质提取物的糖蛋白质组学分析中,Cys-Mal@SiO2共鉴定出1551条糖肽,属于466个糖蛋白的906个N-糖基化位点,比Cys@SiO2和Mal@SiO2鉴定糖肽数、蛋白质数和N-糖基化位点数分别多211、67、127个和289、76、193个.将Cys-Mal@SiO2成功应用于低丰度糖肽的选择性富集与鉴定,在糖蛋白质组学研究中体现出良好的应用潜力.
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