Au-cysteine modified macroporous adsorption resin: preparation and highly selective enrichment and identification of N-linked glycopeptides from the complex biological sample

The development of hydrophilic stationary phase for highly efficient and selective enrichment and identification of low-abundance glycopeptides from a complex biological sample is an important prerequisite in glycoproteomics research. Herein, a hydrophilic cysteine functionalized macroporous adsorption resin/gold nanoparticle (MAR/Au-Cys) was synthesized and applied to the highly selective enrichment and identification of N-linked glycopeptides. The unique porous structure and high specific surface area of MAR provide abundant bonding sites for the immobilization of Au nanoparticles, and subsequently for the loading of cysteine via robust Au-S bonds. The structure and chemical composition of MAR/Au-Cys were confirmed by scanning electron microscopy, nitrogen adsorption-desorption analysis, mercury intrusion porosimetry and Fourier-transform infrared spectroscopy. Besides, the MAR/Au-Cys exhibited good performance in glycopeptide enrichment of tryptic digests from human immunoglobulin G and horseradish peroxidase by operation of a HILIC-SPE tip, revealing good universality, high enrichment selectivity, low detection limit (2.5 fmol) and satisfactory enrichment recovery (above 79%). Furthermore, the proposed material was successfully utilized in the N-glycoproteomics analysis of a complex biological sample. 1305 unique glycopeptides with 883 N-glycosylation sites from 476 different N-linked glycoproteins were identified from the proteins extracted from mouse liver. These results show that the proposed Au-cysteine modified macroporous adsorption resin is a promising affinity material for glycoproteomics research of real complex biological samples