Separation methods hyphenated to mass spectrometry for the characterization of the protein glycosylation at the intact level

Abstract

Glycosylation is one of the most common post-translational modifications of proteins that affects their biological activity, solubility, and half-life. Therefore, its characterization is of great interest in proteomic, particularly from a diagnostic and therapeutic point of view. However, the number and type of glycosylation sites, the degree of site occupancy and the different possible structures of glycans can lead to a very large number of isoforms for a given protein, called glycoforms. The identification of these glycoforms constitutes an important analytical challenge. Indeed, to attempt to characterize all of them, it is necessary to develop efficient separation methods associated with a sensitive and informative detection mode, such as mass spectrometry (MS). Most analytical methods are based on bottom-up proteomics, which consists in the analysis of the protein at the glycopeptides level after its digestion. Even if this approach provides essential information, including the localization and composition of glycans on the protein, it is also characterized by a loss of information on macro-heterogeneity, i.e. the nature of the glycans present on a given glycoform. The analysis of glycoforms at the intact level can overcome this disadvantage. The aim of this review is to detail the state-of-the art of separation methods that can be easily hyphenated with MS for the characterization of protein glycosylation at the intact level. The different electrophoretic and chromatographic approaches are discussed in detail. The miniaturization of these separation methods is also discussed with their potential applications. While the first studies focused on the development and optimization of the separation step to achieve high resolution between isoforms, the recent ones are much more application-oriented, such as clinical diagnosis, quality control, and glycoprotein monitoring in formulations or biological samples.

Keywords: Capillary electrophoresis; Glycosylation; Intact protein; Liquid chromatography; Mass spectrometry.