J. Biochem, 1990, Vol. 108, No. 1 92-98
© 1990 Japanese Biochemical Society
research-article |
High-Performance Liquid Chromatography—Mass Spectrometry of Glycosphingolipids: II. Application to Neutral Glycolipids and Monosialogangliosides1
Department of Membrane Biochemistry, The Tokyo Metropolitan Institute of Medical Science Bunkyo-ku, Tokyo 113
We previously reported a method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of molecular species of GlcCer and IV3 ßGal-Gb4 Cer [M. Suzuki et al. (1989) J. Biochem. 105, 829–833]. In this paper, we report a modification of this HPLC/FAB/MS method, which was used for the separation and characterization of neutral glycosphingolipids (GlcCer, LacCer, Gb3Cer, Gb4Cer, and IV3
GalNAc-Gb4Cer) and monosialogangliosides [GM3(NeuAc or NeuGc), GM2 (NeuAc or NeuGc), and GM1 (NeuAc or NeuGc)]. Mixtures of the purified neutral glycolipids and monosialogangliosides were subjected to HPLC on a silica gel column, with programmed elution with isopropanol-n-hexane-water, with or without ammonium hydroxide. In order to obtain mass spectra and mass chromatograms of individual components, effluent from the HPLC column was mixed with a methanol solution of triethanolamine, which was used as the matrix for the FAB ionization, and one-thirtieth of the effluent mixture was introduced into a mass spectrometer through a frit interface. A mixture of the five neutral glycolipids, 5 µg of each, gave five peaks on a mass chromatogram obtained by monitoring of the corresponding major pseudo-molecular ions. A mixture of the six monosialogangliosides, 5 µg of each, gave six peaks on a mass chromatogram obtained by monitoring of the major pseudo-molecular ions, indicating that GM3, GM2, and GM1 were clearly separated, and that separation due to differences in sialic acid species was also achieved. In the mass spectra of the neutral glycolipids and monosialogangliosides, pseudo-molecular ions and fragment ions due to the elimination of sugar moieties were clearly detected. We applied this method to the analysis of a mixture of neutral glycolipids isolated from human erythrocytes and that of a mixture of monosialogangliosides isolated from the liver of a mouse, GlcCer, LacCer, Gb3Cer, Lc3Cer, Gb4Cer, and nLc4Cer and GM3(NeuAc), GM3(NeuGc), GM2(NeuGc), and GM1(NeuGc) being detected, respectively. This method is of great advantage for the structural characterization of neutral glycolipids and monosialogangliosides, especially when they are in mixtures and/or in small amounts. The method will also be quite promising for other neutral glycolipids and gangliosides with the establishment of suitable separation conditions on HPLC.
1This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, and a grant from the Mitsubishi Foundation.