Analysis of 8-aminonaphthalene-1,3,6-trisulfonic acid labelled N-glycans by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.
Fluorophore-assisted carbohydrate electrophoresis (FACE) is a fast and efficient analytical method which is now widely used in glycobiology for the separation and quantification of free or glycoprotein-released oligosaccharides. However, since identification by FACE of N-glycan structures is only based on their electrophoretic mobility after labelling with 8-aminonaphthalene-1,3, 6-trisulfonic acid (ANTS), co-migration of derived glycans on gel could occur which may result in erroneous structural assignments. As a consequence, a protocol was developed for the fast and efficient matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometric analysis of ANTS-labelled N-glycans. N-Glycans were isolated from plant and mammalian glycoproteins, reductively aminated with the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. The ANTS-labelled glycans were eluted from FACE gel slices and then analysed by MALDI-TOF mass spectrometry in negative ion mode. Using 3-aminoquinoline containing 2.5 mM citrate NH(4)(+) as matrix, neutral N-linked N-glycans, as well as labelled sialylated oligosaccharides, were found to be easily detected in the 2-10 picomole range giving rise to ¿M - H(-) ions.
Lemoine
J
J
Laboratoire de Chimie Biologique, CNRS UMR 8576, Universit¿e de Lille, 59655 Villeneuve d'Ascq, France. jerome.lemoine@univ-lille1.fr
Cabanes-Macheteau
M
M
Bardor
M
M
Michalski
J C
JC
Faye
L
L
Lerouge
P
P
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009281:Naphthalenes; D011134:Polysaccharides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D005456:Fluorescent Dyes; D009281:Naphthalenes; D011134:Polysaccharides; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid