The structures of
acidic glycosphingolipids in
colon adenocarcinoma have been analyzed extensively using a number of conventional methods, such as thin-layer chromatography and methylation analysis, and a variety of
acidic glycosphingolipids present in the tissues have been reported. However, because of a number of limitations in the techniques used in previous studies in terms of resolution, quantification, and sensitivity, we employed a different method that could be applied to small amounts of tissue. In this technique, the
carbohydrate moieties of
acidic glycosphingolipids from approximately 20mg of
colon adenocarcinoma were released by
endoglycoceramidase II and were labeled by pyridylamination. They were separated and structurally characterized by a two-dimensional HPLC mapping technique, electrospray ionization tandem mass spectrometry (ESI-MS/MS), and enzymatic cleavage. A total of 22 major acidic
glycosphingolipid structures were identified, and their relative quantities were revealed in detail. They are composed of 1 sulfated (SM3), 1 lacto-series (SLe(a)), 6 kinds of ganglio-series, and 14 kinds of neolacto-series
glycosphingolipids. They include most of the
acidic glycosphingolipids previously reported to be present in the tissues and two previously unknown
fucogangliosides sharing the same terminal structure: NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, and NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3-Galbeta1-4Glc. Thus, this highly sensitive, high-resolution analysis enabled the identification of novel structures of
acidic glycosphingolipids from small amounts of already comprehensively studied cancerous tissues. This method is a powerful tool for microanalysis of
glycosphingolipid structures from small quantities of cancerous tissues and should be applicable to different types of malignant tissues.