Protein glycosylation analysis with capillary-based electromigrative separation techniques

Abstract

An impressive complexity is associated with glycoproteins due to the microheterogeneity of glycosylation as posttranslational modification giving rise to a vast number of isoforms. The full characterization of glycoproteins is difficult to achieve, and a number of analytical methods have to be combined for a detailed understanding of glycosylation. In this review, we focus on capillary electromigrative separation techniques in the formats capillary electrophoresis, micellar electrokinetic chromatography, and capillary sieving electrophoresis. These separation techniques can be applied to all levels of glycosylation analysis including intact glycoproteins, glycopeptides, and released glycans. We here discuss the separation characteristics for each method and the information that they can provide for each level. Detection issues, especially laser-induced fluorescence detection and mass spectrometry are taken into account. In addition, tables provide an overview on the achievements made from the very beginning of glycosylation research by electromigrative separation techniques. From the literature presented here it is clear, that glycosylation analysis by electromigrative separation techniques is on the edge of transition of basic research and method development towards applications. First proof-ofprinciple studies for in-depth glycoprotein characterization and clinical diagnosis are described. However, this overview also shows that many basic aspects of separation have not yet been fully understood and more research is necessary to be able to fully use the capabilities of electromigrative separation techniques.

Coupling porous sheathless interface MS with transient‐ITP in neutral capillaries for improved sensitivity in glycopeptide analysis

“…Commonly used strategies for mass spectrometric IgG glycosylation profiling in proteolytic digests are RP and hydrophilic interaction LC coupled to MS (RPLC‐MS and HILIC‐MS) and direct mass spectrometric analysis of glycopeptides by MALDI‐MS . In recent years, CE‐MS has been applied for the analysis of glycopeptides from not only IgG but also erythropoietin proteolytic digests .…”

Online enzymatic sequencing of glycans from Trastuzumab by phospholipid‐assisted capillary electrophoresis

“…Several examples of CE separations of glycans accomplished without mass spectrometric detection are described in the literature [33][34][35][36][37]. Most separations are performed under reversed polarity with a covalently modified capillary to suppress electroosmotic flow.…”

Capillary zone electrophoresis for the analysis of glycoforms of cellobiohydrolase

“…Furthermore, these techniques are equipment intensive and require specialised skills. Therefore, several studies have been reported on the use of CZE with optical detection methods for routine analysis of glycoforms due to its high resolution, sensitivity, reduced analysis time and ease-of-use [23][24][25]. Taverna et al [26] propose that a strategy for CZE analysis of glycoconjugates is to try electrolytes at the pH extremes, to ensure that at least a single peak can be identified, followed by testing pHs closer to the isoelectric point of the glycoprotein to improve glycoform separation.…”