An ultrafast and highly efficient enrichment method for both N-Glycopeptides and N-Glycans by bacterial cellulose

Abstract

A powerful and fast glycopeptide/glycan enrichment method is critical for the efficiency and throughput of mass spectrometry (MS)-based glycoproteomic and glycomic analyses, especially for large-scale sample analysis. Here, we report an ultrafast and effective method for both intact N-glycopeptide and N-glycan enrichment and apply it to human serum samples. In this method, a natural hydrophilic material, bacterial cellulose (BC), was adopted and fully optimized for enrichment. This method offers the following advantages: (i) The enrichment material has natural hydrophilicity and is low-cost, biocompatible, biodegradable and easily accessible; (ii) the whole enrichment procedure is remarkably simple and fast. It takes only 10 min for intact glycopeptides/glycans to be easily purified from mixtures; (iii) the specificity of this method is over 94% for both glycan and glycopeptide enrichment; and (iv) the outstanding specificity of this technique enables high isolation efficiency for the enrichment of both intact glycopeptides and glycans. A total of 36 N-glycans and 31 N-glycopeptides were identified from human immunoglobulin G (IgG). The glycan and glycopeptide absorption capacity of BC was as high as 333 μg/mg and 250 μg/mg (IgG/BC) respectively. The selectivity for glycan and glycopeptide enrichment reached 1:100 (IgG/bovine serum albumin (BSA), molar ratio) and 1:200 (maltoheptaose (DP7)/BSA, molar ratio), respectively. Furthermore, a total of 159 N-glycans and 523 N-glycopeptides were identified in human serum by using this method. Overall, the BC-based enrichment method we present here provides an ultrafast and highly efficient method for the enrichment of both N-glycopeptides and N-glycans in complex samples and shows great potential in large-scale glycoproteomic and glycomic analyses.