ABSTRACT
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.
Subject(s)
Aminopyridines/chemistry , Ethylamines/chemistry , Fluorescent Dyes/chemistry , Gangliosides/analysis , N-Acetylneuraminic Acid/chemistry , Oligosaccharides/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Chromatography, High Pressure Liquid , Fluorescence , G(M3) Ganglioside/analysis , Lactose/analogs & derivatives , Lactose/analysisABSTRACT
The anti-viral activity of gangliosides such as SPG (sialylparagloboside), GD1a, GM3, and GM4 was assessed by inhibition of the cytopathy of MDCK cells due to infection with the influenza virus A/PR/8/34. The inhibitory effect was in the following sequence: SPG>GD1a>GM3>GM4. The IC50 of SPG and GD1a was 7 and 70 microM, respectively, indicating that they are more effective than the representative inhibitor amantadine. Although 3'-sialyllactose (3'-SL) and 3'-sialyllactosamine (3'-SLN), which are identical to the terminal trisaccharides of GM3 and SPG, respectively, did not show any inhibitory effect, introduction of an amino group to the reducing end of 3'-SL following amidation with lauroyl chloride gave the inhibitory potency, which was comparable to that of GM3. These results suggest that the viral hemagglutinin recognizes exogenous sialyloligosaccharides rather than inherent sialyloligosaccharides expressed on MDCK cells, since introduction of the hydrophobic moiety to oligosaccharides might cause micelle formation.
