Analysis of 8-aminonaphthalene-1,3,6-trisulfonic acid labelled N-glycans by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

✍ Scribed by Jérôme Lemoine; Marion Cabanes-Macheteau; Muriel Bardor; Jean-Claude Michalski; Loïc Faye; Patrice Lerouge

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 98 KB
• Volume: 14
• Category: Article
• ISSN: 0951-4198
• DOI: 10.1002/(sici)1097-0231(20000130)14:2<100::aid-rcm845>3.0.co;2-w

✦ Synopsis

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), comigration 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 À H] À ions.