Simultaneous Release and Labeling of O- and N‑Glycans Allowing for Rapid Glycomic Analysis by Online LC-UV-ESI-MS/MS

Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herei...

Full description

Saved in:
Bibliographic Details
Published in:Journal of proteome research 2018-07, Vol.17 (7), p.2345-2357
Main Authors: Wang, Chengjian, Lu, Yu, Han, Jianli, Jin, Wanjun, Li, Lingmei, Zhang, Ying, Song, Xuezheng, Huang, Linjuan, Wang, Zhongfu
Format: Article
Language:English
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization. In this procedure, glycoproteins are incubated with 1-phenyl-3-methyl-5-pyrazolone (PMP) in aqueous ammonium hydroxide, making O-glycans released from protein backbones by β-elimination and N-glycans liberated by alkaline hydrolysis. The released glycans are promptly derivatized with PMP in situ by Knoevenagel condensation and Michael addition, with peeling degradation almost completely prevented. The recovered mixture of O- and N-glycans as bis-PMP derivatives features strong ultraviolet (UV) absorbing ability and hydrophobicity, allowing for high-resolution chromatographic separation and high-sensitivity spectrometric detection. Using this technique, O- and N-glycans were simultaneously prepared from some model glycoproteins and complex biological samples, without significant peeling, desialylation, deacetylation, desulfation or other side-reactions, and then comprehensively analyzed by online HILIC-UV-ESI-MS/MS and RP-HPLC-UV-ESI-MS/MS, with which some novel O- and N-glycan structures were first found. This method provides a simple, versatile strategy for high-throughput glycomics analysis.
ISSN:1535-3893
1535-3907
DOI:10.1021/acs.jproteome.8b00038