Reliable N-Glycan Analysis–Removal of Frequently Occurring Oligosaccharide Impurities by Enzymatic Degradation

Abstract

Glycosylation, especially N-glycosylation, is one of the most common protein modifications, with immense importance at the molecular, cellular, and organismal level. Thus, accurate and reliable N-glycan analysis is essential in many areas of pharmaceutical and food industry, medicine, and science. However, due to the complexity of the cellular glycosylation process, in-depth glycoanalysis is still a highly challenging endeavor. Contamination of samples with oligosaccharide impurities (OSIs), typically linear glucose homo-oligomers, can cause further complications. Due to their physicochemical similarity to N-glycans, OSIs produce potentially overlapping signals, which can remain unnoticed. If recognized, suspected OSI signals are usually excluded in data evaluation. However, in both cases, interpretation of results can be impaired. Alternatively, sample preparation can be repeated to include an OSI removal step from samples. However, this significantly increases sample amount, time, and effort necessary. To overcome these issues, we investigated the option to enzymatically degrade and thereby remove interfering OSIs as a final sample preparation step. Therefore, we screened ten commercially available enzymes concerning their potential to efficiently degrade maltodextrins and dextrans as most frequently found OSIs. Of these enzymes, only dextranase from Chaetomium erraticum and glucoamylase P from Hormoconis resinae enabled a degradation of OSIs within only 30 min that is free of side reactions with N-glycans. Finally, we applied the straightforward enzymatic degradation of OSIs to N-glycan samples derived from different standard glycoproteins and various stem cell lysates.