Da. Wiesner et Cc. Sweeley, MICROSCALE ANALYSIS OF GLYCOSPHINGOLIPIDS BY METHANOLYSIS, PERACETYLATION, AND GAS-CHROMATOGRAPHY, Analytical biochemistry, 217(2), 1994, pp. 316-322
A method for the analysis of pure samples of individual glycosphingoli
pids by microscale methanolysis, peracetylation, and gas chromatograph
y is described. Solvolysis of glycosphingolipids in dry methanolic HCl
and peracetylation were conducted in a single 4.5-cm sealed capillary
tube (2 mm i.d.), after which the products were directly injected int
o a gas chromatograph. Total-component analysis (i.e., analysis of the
sugar, fatty acid, and sphingosine moieties) was possible after a 45-
min chromatographic run. Time-course studies of the acid-catalyzed met
hanolysis of Gal beta 1-4GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4G
lc beta 1-1Cer ganglioside at 80, 110, and 150 degrees C showed that m
ethanolysis was complete after 2 h at 110 degrees C. Rates of methanol
ysis of individual components were compared and the release of the fat
ty acid moiety from the long-chain base was shown to be the slowest re
action. The methanolysis of all glycosidic bonds was complete in 0.5 h
. Peracetylated methanolysis products were very stable over time and p
rovided for good gas chromatographic detection of subnanomolar amounts
of hexose, hexosamine, fatty acid, sialic acid, and long-chain sphing
oid base components. Recoveries of fucose and N-acetylglucosamine were
determined with reference samples of Fuc alpha 1-2Lac and lacto-N-fuc
osylpentaose II. Applications of the method are presented for the comp
onent analysis of a gift mixture of NeuAc alpha 2-6Gal beta 1-4GlcNAc
beta 1- Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-1 Cer gan
glioside and NeuAc alpha 2-6Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1-
3Gal beta 1-4(Fuc alpha 1-3) GlcNAc beta 1-3Gal beta 1-4Glc-Cer gangli
oside and analysis of NeuAc alpha 2-3GalB1-4GlcB1-1 Cer isolated from
human plasma. (C) 1994 Academic Press,Inc.