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.

Figure 1

Six representative structures of d conformer monosaccharides. Glucose, mannose, and galactose are common unsubstituted hexose saccharides. Fucose has one less hydroxyl group and is termed a deoxy-hexose sugar. N-Acetylglucosamine and N-acetylneuraminic acid represent substituted hexose saccharides.

Figure 2

Two structures of sialyllactose that differ only by the linkage between the sialic acid and galactose monosaccharides. The structure in panel A is 6′-sialyllactose, which is composed of an α2–6 linkage. The structure in panel B is 3′-sialyllactose, which is composed of an α2–3 linkage.

Figure 3

Depiction of an N-glycan, which is composed of several monosaccharides, removed from an antibody. The reducing end (B), which is located where the N-acetylglucosamine is cleaved from the antibody, is the target for the attachment of fluorophores, such as APTS.

Figure 4

Simplified mechanism of reductive amination as it is used to label carbohydrates with APTS using a reducing agent (sodium cyanoborohydride).

Figure 5

Basic diagram of the capillary electrophoresis system commonly used to separate APTS-labeled glycans. Separations are performed in reverse polarity (cathode to anode) under suppressed electroosmotic flow using a coated capillary. APTS-labeled glycans are separated by charge-to-size ratio in the order of ascending hydrodynamic volume as shown in the inset and quantified using an electropherogram generated by laser-induced fluorescence.

Figure 6

Electropherogram of three internal standards (DP2, DP3, and DP15 are maltose, maltotriose, and maltopentadecaose, respectively) and APTS labeled N-linked glycan released from human immunoglobulin G in the upper trace. The representative electropherogram of maltooligomers in the lower trace is aligned with DP2, DP3, and DP15 in the upper panel. Reprinted with permission from ref (85). Copyright 2016 American Chemical Society.

Figure 7

Conceptual diagrams of online enzyme and lectin reactions in-capillary. Panel A depicts a galactosylated triantennary glycan separated in the absence and presence of galactosidase. Upon cleavage of the terminal galactose residues, the glycan migrates faster due to mass loss. Panel B depicts galactosylated triantennary glycan in the absence and presence of Erythrina cristagalli lectin. Upon binding to the terminal galactose residues, the glycan is not detected.

Figure 8

An instrument setup for off-line coupling of capillary electrophoresis and matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). The capillary electrophoresis instrument was modified to incorporate the automatic spotting device to deposit the capillary eluent to the MALDI plate from the outlet vial of the capillary electrophoresis instrument, and the matrix required for MALDI detection was delivered from the inlet vial. From ref (101). Copyright 2014 by John Wiley Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.

Figure 9

A cross sectional view of a sheathless CE-ESI MS interface including a porous tip in the front of the capillary outlet immersed in the background electrolyte (BGE) inside the stainless steel cylinder. Reprinted with permission from ref (106). Copyright 2010 American Chemical Society.

Figure 10

MS/MS spectrum of two glycans that overlapped in the capillary electrophoresis separation. The red line in the spectrum shows the signal (m/z) of the precursor ions selected for MS² scan. The observed Y-type fragment ions in the MS/MS spectrum indicate the identity of the monosaccharide that is lost. Reprinted from ref (104), Copyright 2017, with permission from Elsevier.

Figure 11

A representative MS/MS spectrum of a tandem mass tag (TMT) labeled glycan (m/z 779.8). The fragmentation of the TMT labeled glycan is depicted in the structure above the spectrum. The reporter ions, used for quantification, are observed in the lower mass range of the spectrum. Reprinted with permission from ref (105). Copyright 2015 American Chemical Society.

Figure 12

Schematic of heparin disaccharide. The repeating disaccharide is composed of l-iduronic acid and d-glucosamine joined by an α1–4 glycosidic linkage. The R group can be either sulfate or acetate and R′ and R″ can be either sulfate or hydrogen.

Figure 13

Photograph of the laboratory set up for the Biomek FXP Laboratory Automation Workstation, which was used to automate work flow for APTS labeling and purification of carbohydrates. The lab materials required for this include a lid for the pipet box to reduce evaporation (1), a 96-sample tray for a capillary electrophoresis instrument (2), a Peltier shaker (3), 20 μL pipet tips (4), labeling reagents and magnetic beads (5), a 96-well PCR plate on a magnetic stand (6), 1000 μL pipet tips (7), and a 24-well plate for large volumes of reagent (8). From ref (64). Copyright 2016 by SAGE Publications, Reprinted by Permission of SAGE Publications, Inc.

Figure 14

Schematic of a commercially available microfluidic chip for capillary electrophoresis–electrospray ionization–mass spectrometry. The chip consists of sample reservoir S, background electrolyte reservoir B, electrospray ionization pump P, and sample waste SW. The nanospray interface is integrated at the corner of the chip. Adapted with permission from ref (107). Copyright 2017 American Chemical Society.