Chip‐based CE for rapid separation of 8‐aminopyrene‐1,3,6‐trisulfonic acid (APTS) derivatized glycans

Abstract

Fluorescently labeled carbohydrates released from glycoproteins were separated using a commercially available microfluidic chip electrophoresis system. While the instrumentation was primarily designed for DNA analysis it was found that the application base can be easily expanded using the development software provided by the manufacturer. The carbohydrates were released by enzymatic digestion (PNGase F) from glycoproteins present in human plasma after boronic acid - lectin affinity enrichment. After fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonic acid the carbohydrates were separated based on capillary gel electrophoresis mechanism and detected by a fluorescence detector using a blue (470 nm) LED. The separation was completed in 40 s in a microfluidic channel of 14 mm length. Glucose ladder carbohydrate oligomers differing by one glucose unit were baseline separated up to a 20-mer with the main limitation being the detection sensitivity. As expected, the observed resolution in these experiments did not approach that of standard CE with 20 times longer separation distance; however, the chip-based analysis excelled in the speed of the separation. Similar electrophoretic profiles of glycans released from plasma glycoproteins were obtained using a standard CE equipment with 35 cm separation length and microfluidic chips with a separation distance of only 14 mm.

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

“…Capillary electrophoresis separations can be adapted to microfluidics devices, and chip-based electrophoretic separations have been described in literature [42,[44][45][46][47]. Bench-top exoglycosidase sequencing has been used in conjunction with CE to assign glycan structure [48,49].…”

Capillary Electrophoresis/Mass Spectrometry of APTS-Labeled Glycans for the Identification of Unknown Glycan Species in Capillary Electrophoresis/Laser-Induced Fluorescence Systems

“…Different systems based on pseudo gel electrolytes have been reported and applied successfully in high throughput analysis in the pharmaceutical and food industry 14 as well as in clinical screening 12,15 . The detection with fluorescence requires sample pretreatment with fluorescence markers, being 8-aminopyrene-1,3,6-trisulfonic acid (APTS) one of the most applied labels for glycans 16,17 . Standard CSE/LIF approaches for APTS-labeled glycan analysis were performed applying a negative potential to the capillary inlet.…”

Potential of Capillary Electrophoresis (CE) and Chip-CE with Dual Detection (Capacitively-Coupled Contactless Conductivity Detection (C4D) and Fluorescence Detection) for Monitoring of Nicotine and Cotinine Derivatization

“…[26][27][28] Agilent Bioanalyzer, a chip-CE instrument, is commercially available and its potential as a generic chip CE platform has been demonstrated when operated in research mode with access to script editor for fully flexible method editing. 29,30 Applications of this approach included profiling 8-aminopyrene-1,3,6-trisulfonic acid (APTS) derivatized glycans 28 and in-field analysis of derivatized amphetamines. 30 This work investigated potentials of CE with dual detectors (C 4 D and FD) for monitoring of nicotine and cotinine derivatization with aniline to form fluorescent products.…”