Capillary Electrophoresis Separations of Glycans

Abstract

Capillary electrophoresis has emerged as a powerful approach for carbohydrate analyses since 2014. The method provides high resolution capable of separating carbohydrates by charge-to-size ratio. Principle applications are heavily focused on N-glycans, which are highly relevant to biological therapeutics and biomarker research. Advances in techniques used for N-glycan structural identification include migration time indexing and exoglycosidase and lectin profiling, as well as mass spectrometry. Capillary electrophoresis methods have been developed that are capable of separating glycans with the same monosaccharide sequence but different positional isomers, as well as determining whether monosaccharides composing a glycan are alpha or beta linked. Significant applications of capillary electrophoresis to the analyses of N-glycans in biomarker discovery and biological therapeutics are emphasized with a brief discussion included on carbohydrate analyses of glycosaminoglycans and mono-, di-, and oligosaccharides relevant to food and plant products. Innovative, emerging techniques in the field are highlighted and the future direction of the technology is projected based on the significant contributions of capillary electrophoresis to glycoscience from 2014 to the present as discussed in this review.

High‐performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements

“…A classical electrophoresis method such as agarose gel could be used to qualitative separate intact HA chains from the CS contained in FS, by detecting with toluidine blue followed by "stains all" method, but it could not separate intact chains of KS that co-migrate with the CS polysaccharide [17,35]. Capillary electrophoresis could be a valid quantitative alternative as it has been already widely employed for the determination of GAGs, on the basis of their disaccharide composition after enzymatic digestions [5,36,37] but also as intact chains [22][23][24][25][26][27][28][29][30][31], as well as for the determination of their microbial homologues [38,39]. In particular, various mixed solutions of intact HP, CS, DS, HS, HA, and/or OSCS chains, in diverse compositions, have been separated so far by using different HPCE methods that employed a reverse polarity mode, with voltages between −14 kV and −30 kV and temperatures between 15°C and 37°C, mainly employing sodium phosphate or Tris buffers with pH values between 3.0 and 3.5 and UV detection between 200 and 230 nm [5,[21][22][23][24][25][26][27][28].…”

Negatively Charged Yellow‐Emitting 1‐Aminopyrene Dyes for Reductive Amination and Fluorescence Detection of Glycans

“…Therefore, dyes 6 ‐H and 16 with six negative charges may reveal “heavy” glycans undetectable with APTS due to very long retention times caused by the relatively low charge (−3) and the limited brightness. Access to the DNA sequencer with a CGE‐LIF unit will make it possible to evaluate the crosstalk between the emission signals of APTS, on one hand, and the dyes 6 ‐H and 16 , on the other hand (also in conjugates), and their applicability for calibration of the retention times in CGE …”

Current Trends in the Analysis of Post-translational Modifications

“…The derivatives can be subsequently separated by a number of methods, such as HILIC and CE prior to mass spectrometric analysis. CE is capable of separating positional isomers as well as differently linked glycans with high resolution and short analysis time, while HILIC tends to yield better retention time repeatability [89,90]. It is also demonstrated that the techniques show notable complementarity in the glycoform profiling of therapeutic proteins [91].…”