Centrifugation Assisted Microreactor Enables Facile Integration of Trypsin Digestion, Hydrophilic Interaction Chromatography Enrichment, and On-Column Deglycosylation for Rapid and Sensitive N-Glycoproteome Analysis

Abstract

Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.

Highly Efficient Identification of O‐GalNAc Glycosylation by an Acid‐Assisted Glycoform Simplification Approach

“…Enrichment and Simplification of O‐GalNAc Glycopeptides : The O‐GalNAc glycopeptides were enriched by using HILIC tip materials according to our previous reports . The digestions of proteins were re‐dissolved in the loading buffer (80% ACN/1% TFA).…”

“…In the analysis of N‐glycoproteome, N‐glycopeptides were typically subjected to PNGase F treatment to release glycans, which could effectively decrease the microheterogeneity of glycopeptides, as well as enhance the detection sensitivity of N‐linked glycopeptides . Yet, there was no such glycosidase that could release O‐glycans with high efficiency.…”

Mapping the N-linked glycosites of rice (Oryza sativa L.) germinating embryos

“…The HILIC enrichment of N- glycopeptides was processed as described with minor modifications [37,38]. Briefly, the peptide fractions were equilibrated in 40 μL loading buffer (80% acetonitrile/1% trifluoroacetic acid), then pipetted into a tip containing HILIC beads (4 μm, 100 Å) (Dalian Institute of Chemical Physics, Dalian, China).…”

Comprehensive quantification of N-glycoproteome in Fusarium graminearum reveals intensive glycosylation changes against fungicide

“…for glycopeptide enrichment and they identified 4,783 N-glycosylation sites in a single human tissue [13,14]. The HILIC-based method benefits for its simple operation, low sample cost and high efficiency compared with lectin affinity and boronate affinity methods, and was adopted in this work.…”

“…The HILIC enrichment of N-glycopeptides were performed according to previous reports with minor modifications [13,14]. The peptide fractions were first redissolved in the enrichment loading buffer (80% ACN/1% TFA) and then pipetted into a HILIC tip.…”