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Titolo:
Ultrasensitive profiling and sequencing of N-linked oligosaccharides usingstandard DNA-sequencing equipment
Autore:
Callewaert, N; Geysens, S; Molemans, P; Contreras, R;
Indirizzi:
State Univ Ghent, Dept Mol Biol, Unit Fundamental & Appl Mol Biol, B-9000 Ghent, Belgium State Univ Ghent Ghent Belgium B-9000 pl Mol Biol, B-9000 Ghent, Belgium Flanders Interuniv Inst Biotechnol, B-9000 Ghent, Belgium Flanders Interuniv Inst Biotechnol Ghent Belgium B-9000 0 Ghent, Belgium
Titolo Testata:
GLYCOBIOLOGY
fascicolo: 4, volume: 11, anno: 2001,
pagine: 275 - 281
SICI:
0959-6658(200104)11:4<275:UPASON>2.0.ZU;2-L
Fonte:
ISI
Lingua:
ENG
Soggetto:
DESORPTION/IONIZATION MASS-SPECTROMETRY; CAPILLARY GEL-ELECTROPHORESIS; LASER-INDUCED FLUORESCENCE; FLUOROMETRIC DETECTION; CHROMATOGRAPHY; GLYCOPROTEINS; MIXTURES; RELEASE;
Keywords:
alpha(1)-acid glycoprotein; APTS; DNA sequencer; MALDI-TOF-MS; N-glycan;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
19
Recensione:
Indirizzi per estratti:
Indirizzo: Contreras, R State Univ Ghent, Dept Mol Biol, Unit Fundamental & Appl Mol Biol, KL Ledeganckstr 35, B-9000 Ghent, Belgium State Univ Ghent KL Ledeganckstr 35 Ghent Belgium B-9000 ium
Citazione:
N. Callewaert et al., "Ultrasensitive profiling and sequencing of N-linked oligosaccharides usingstandard DNA-sequencing equipment", GLYCOBIOLOG, 11(4), 2001, pp. 275-281

Abstract

The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycoprotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a challenging task. We present here methodology allowing the medium-throughput analysis of N-glycans derived from low picomole amounts of glycoproteins using the standard DNA-sequencing equipment available in any life sciences laboratory, The high sensitivity of the overall analytical process (from glycoprotein to results) is obtained using state-of-the-art deglycosylation procedures combined with a highly efficient and reproducible novel postderivatization cleanup step involving Sephadex G10 packed 96-well filterplates, Allsample preparation steps (enzymatic deglycosylation with PNGase F, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderivatization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresis and MALDI-TOF-MS platforms already in use in some glycobiology labs and anticipates the higher throughput that will be offered by the capillary-array-based DNA sequencers currently penetrating the market. The described technology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/04/20 alle ore 08:33:56