Preparation of cysteine-click maltose modified silica as a hydrophilic interaction liquid chromatography material for the enrichment of glycopeptides

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.