Analysis of protein glycosylation by mass spectrometry.

Abstract

There is a growing pharmaceutical market for protein-based drugs for use in therapy and diagnosis. The rapid developments in molecular and cell biology have resulted in production of expression systems for manufacturing of recombinant proteins and monoclonal antibodies. These proteins are glycosylated when expressed in cell systems with glycosylation ability. For glycoproteins intended for therapeutic administration it is important to have knowledge about the structure of the carbohydrate side chains to avoid cell systems that produce structures, which in humans can cause undesired reactions, e.g., immunological and unfavorable serum clearance rate. Structural analysis of glycoprotein oligosaccharides requires sophisticated instruments like mass spectrometers and nuclear magnetic resonance spectrometers. However, before the structural analysis can be conducted, the carbohydrate chains have to be released from the protein and purified to homogeneity, and this is often the most time-consuming step. Mass spectrometry has played and still plays an important role in analysis of protein glycosylation. The superior sensitivity compared to other spectroscopic methods is its main asset. Structural analysis of carbohydrates faces several problems, however, due to the chemical nature of the constituent monosaccharide residues. For oligosaccharides or glycoconjugates, the structural information from mass spectrometry is essentially limited to monosaccharide sequence, molecular weight, an only in exceptional cases glycosidic linkage positions can be obtained. In order to completely establish an oligosaccharide structure, several other structural parameters have to be determined, e.g., linkage positions, anomeric configuration and identification of the monosaccharide building blocks. One way to address some of these problems is to work on chemical pretreatment of the glycoconjugate, to specifically modify the carbohydrate chain. In order to introduce specific modifications, we have used periodate oxidation and trifluoroacetolysis with the objective of determining glycosidic linkage positions by mass spectrometry.