A Rapid Procedure for the Purification of 8-Aminopyrene Trisulfonate (APTS)-Labeled Glycans for Capillary Electrophoresis (CE)-Based Enzyme Assays

Abstract

Purified glycan standards are required for glycan arrays, characterizing substrate specificities of glycan-active enzymes, and to serve as retention-time or mobility standards for various separation techniques. This chapter describes a method for the rapid separation, and subsequent desalting, of glycans labeled with the highly fluorescent fluorophore 8-aminopyrene 1,3,6-trisulfonate (APTS). By using fluorophore-assisted carbohydrate electrophoresis (FACE) on polyacrylamide gels, which utilizes equipment readily available in most molecular biology laboratories, many APTS-labeled glycans can be simultaneously resolved. Excising specific gel bands containing the desired APTS-labeled glycans, followed by glycan elution from the gel and subsequent solid-phase extraction (SPE), yields single glycan species free of excess labeling reagents and buffer components. This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples.

Capillary Electrophoresis Analysis of Bovine Milk Oligosaccharides Permits an Assessment of the Influence of Diet and the Discovery of Nine Abundant Sulfated Analogues

“…CE-LIF has proven to be an ideal separation technique for highly polar and/or charged analytes like oligosaccharides, having previously been used to characterize HMOs and porcine or equine milk oligosaccharides. Prior to CE analysis, oligosaccharides are typically labeled at their reducing ends with the highly fluorescent and charged compound APTS ,, (Figure S3), enabling the efficient separation of both neutral and acidic oligosaccharides and ensuring equivalent response factors across all oligosaccharide structures. Here, CE-LIF was used as a screening tool to rapidly profile the intact BMOs (Figure ).…”

“…The supernatant was dried in vacuo using a SpeedVac Concentrator (Thermo-Fisher Scientific) at ambient temperature. Further BMO purification, including the elimination of the majority of the lactose present (Table S1), was performed using preconditioned graphitized carbon SPE cartridges (Supelco, ENVICarb, 200 mg). Acidic (50% ACN/0.1% TFA) and neutral (20% ACN) BMO-containing SPE fractions were combined and dried in vacuo .…”

“…Unless otherwise noted, 18 MΩ water was provided by a Barnstead E-Pure water purification system (Thermo Fisher Scientific, Waltham, MA, USA) for preparing all aqueous solutions, including uHPLC mobile phases. 8-Aminopyrene-1,3,6-trisulfonic acid (APTS) was synthesized exactly according to a published procedure 25 with the exception that the total reaction time was reduced from 30 to 3 h. APTS was further purified over a graphite column 26 and quantified by fluorescence, using commercially available APTS (SCIEX, Washington, DC, USA) as a calibration standard. APTS was stored as a 100 mM stock in 0.9 M citric acid at −20 °C.…”

“…30 (4) BMOs were labeled with APTS (16 h, room temperature) exactly as previously described (Figure S3). 25,26 Labeling reactions were quenched by adding 100 μL of H 2 O before all samples were analyzed by CE-LIF using reversed polarity (−30 kV) and a NCHO separation buffer (SCIEX) as the background electrolyte. All peaks not attributable to the APTS reagents were manually integrated using 32 Karat (version 8.0) software.…”

Capillary Electrophoresis Separations of Glycans

“… 70 Finally, slab gel electrophoresis was reported for glycan purification by excising gels and extracting the labeled glycans. 51 …”