Structural Analyses of Carbohydrate Moieties of Glycoproteins by Microwave-Assisted Partial Acid Hydrolysis and Mass Spectrometry

Bao-Shiang      Lee; Sangeeth      Krishnanchettiar; G.   D.   Lasanthi P. Jayathilaka; Syed   Salman   Lateef; Shalini      Gupta

Abstract

Characterization of carbohydrate moieties of glycoproteins using microwave-assisted partial acid hydrolysis (MAPAH) and mass spectrometry (MS) are described in this review. Acids including hydrochloric acid (HCl), trifluoroacetic acid (TFA), and phosphoric acid (H3PO4) can be used to induce partial hydrolysis of the carbohydrate moieties of glycoproteins in as short as 30 s of microwave exposure. High resolving power of MS allows distinction between different glycopeptide fragments. Several glycoproteins including bovine pancreatic ribonuclease B (RNase B, MW ∼15 kDa), egg white avidin (MW ∼16 kDa), human serum α1-acid glycoprotein (α1-AGP, MW ∼36 kDa), horseradish peroxidase (HRP, MW ∼44 kDa), fetal calf serum fetuin (BSF, MW ∼45 kDa), and glucose oxidase from Aspergillus niger (GO, MW ∼75 kDa), are used to demonstrate this technique. Information of the sugar composition and/or arrangement of the carbohydrate moieties can be readily obtained either from glycoproteins directly or digested glycopeptides of the glycoproteins. This method is fast, sensitive, and a reliable technique. Challenges when working on larger and heavily glycosylated glycoproteins are discussed.

Keywords: Glycoproteins, Glycopeptides, Carbohydrate, Posttranslational modification (PTM), Microwave-assisted partial, acid hydrolysis (MAPAH), Glasstip static probe, Nanospray, MALDI-TOF MS, FT-ICR MS