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

Journal of Proteome Research
Chengjian WangZhongfu Wang

Abstract

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 a...Continue Reading

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Citations

Jan 29, 2021·Current Opinion in Structural Biology·Tomislav ČavalSergey Y Vakhrushev
Sep 7, 2019·Journal of Agricultural and Food Chemistry·Yu LuZhongfu Wang

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