Chip-based CE for rapid separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS) derivatized glycans.
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.
Irish Separation Science Cluster, National Centre for Sensor Research and School of Chemical Sciences, Dublin City University, Dublin, Ireland. smejkal@iach.cz
Petr
P
Smejkal
Akos
A
Szekrényes
Markéta
M
Ryvolová
František
F
Foret
András
A
Guttman
Fritz
F
Bek
Mirek
M
Macka
D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011721:Pyrenes / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013997:Time Factors
C098210:1-aminopyrene-3,6,8-trisulfonic acid; D006023:Glycoproteins; D009844:Oligosaccharides; D011721:Pyrenes; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase