Fluorescent labeling of a carboxyl group of sialic acid for MALDI-MS analysis of sialyloligosaccharides and ganglioside

• Published in: Biochemical and biophysical research communications 2009-01, Vol.378 (4), p.890-894
• Main Authors: Endo, Shin-ichi, Morita, Minoru, Ueno, Masaki, Maeda, Tadakazu, Terabayashi, Takashi
• Format: Article
• Language: English
• Subjects: Amidation; Aminopyridines - chemistry; Chromatography, High Pressure Liquid; Ethylamines - chemistry; Fluorescence; Fluorescent Dyes - chemistry; Fluorescent labeling; G(M3) Ganglioside - analysis; Ganglioside 2-(2-pyridilamino)ethylamine; Gangliosides - analysis; Lactose - analogs & derivatives; Lactose - analysis; MALDI-TOF-MS; N-Acetylneuraminic Acid - chemistry; Oligosaccharides - analysis; Sialic acid; Sialylglycoconjugate; Sialyloligosaccharide; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2008.12.011

We developed a modified method enabling stable MALDI-MS analysis and fluorescent detection of sialyl-compounds. The modification involved the amidation of sialic acid (Neu5Ac) at the position of the carboxyl group using the fluorescent reagent, 2-(2-pyridilamino)ethylamine (PAEA). In this study the following sialyl-compounds were amidated, 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), and ganglioside GM3. Yields of PAEA-3′-SL, PAEA-6′-SL, and PAEA-GM3 were 45%, 60%, and 30%, respectively. The PAEA-amidation enabled fluorescence detection of structural isomers using HPLC and TLC at sensitivity levels as low as pmol. In MALDI-TOF-MS and/or MS/MS analysis in positive ion mode, PAEA-amidation provided the following advantages: suppression of preferential cleavage of Neu5Ac; enhancement of molecular-related ion intensities; simplification of MS spectra; and finally, since PAEA-amidation did not cleave the linkage between sugar and aglycon of sialylglycoconjugate, MALDI-TOF-MS and MS/MS analyses revealed the complete structure of the molecule.