JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2001; 36: 21–29 Nano-electrospray ionization time-of-flight mass spectrometry of gangliosides from human brain tissue Wolfgang Metelmann, ˇ Zeljka Vukeli ´ c † and Jasna Peter-Katalini ´ c * Laboratory for Biomedical Analysis, Institute for Medical Physics and Biophysics, University of M ¨ unster, Robert-Koch-Str. 31, D-48149 M ¨ unster, Germany Received 31 May 2000; Accepted 26 September 2000 A general approach for the detection and structural elucidation of brain ganglioside species GM1, GD1 and GT1 by nano-electrospray ionization quadrupole time-of-flight (nanoESI-QTOF) mass spectrometry (MS), using combined data from MS and MS/MS analysis of isolated native ganglioside fractions in negative ion mode and their permethylated counterparts in the positive ion mode is presented. This approach was designed to detect and sequence gangliosides present in preparatively isolated ganglioside fractions from pathological brain samples available in only very limited amounts. In these fractions mixtures of homologue and isobaric structures are present, depending on the ceramide composition and the position of the sialic acid attachment site. The interpretation of data for the entire sequence, derived from A, B, C and Y ions by nanoESI-QTOFMS/MS in the negative ion mode of native fractions, can be compromized by ions arising from double and triple internal cleavages. To distinguish between isobaric carbohydrate structures in gangliosides, such as monosialogangliosides GM1a and GM1b, disialogangliosides GD1a, GD1b and GD1c or trisialogangliosides GT1b, GT1c and GT1d, the samples were analysed after permethylation in the positive ion nanoESI-QTOFMS/MS mode, providing set of data, which allows a clear distinction for assignment of outer and inner fragment ions according to their m/z values. The fragmentation patterns from native gangliosides obtained by low-energy collision induced dissociation (CID) by nanoESI-QTOF show common behaviour and follow inherent rules. The combined set of data from the negative and positive ion mode low-energy CID can serve for the detection of structural isomers in mixtures, and to trace new, not previously detected, components. Copyright  2001 John Wiley & Sons, Ltd. KEYWORDS: human brain gangliosides; GM1, GD1 and GT1; nano-electrospray ionization quadrupole time-of-flight mass spectrometry; low-energy collision-induced dissociation; native and permethylated gangliosides INTRODUCTION Gangliosides, the glycosphingolipids (GSLs) containing one or more sialic acid residues, are known to occur in all eukary- otic cells, primarily as plasma-membrane components. Their hydrophobic ceramide portion, i.e. N-fatty acyl derivative of a long-chain sphingoid base, is embedded in the outer leaflet Abbreviations: Gangliosides and their precursor glycosphingolipids are abbreviated according to the system of L. Svennerholm (J. Neuro-chem. 1963; 10: 613 and Adv. Exp. Med. Biol. 1980; 125: 11) and the recommendations of the IUPAC–IUB Commission on Biochemical Nomenclature (Eur. J. Biochem. 1977; 79: 11 and Eur. J. Biochem. 1998; 257: 293) as follows: LacCer, Galˇ4Glcˇ1Cer; Gg 4 Cer, Galˇ3GalN-Acˇ4Galˇ4Glcˇ1Cer; nLc 4 Cer, Galˇ4GlcNAcˇ3- Galˇ4Glcˇ1Cer; GM1a or GM1, II 3 -˛-Neu5Ac-Gg 4 Cer; GM1b, IV 3 -˛-Neu5Ac-Gg 4 Cer; GD1a, IV 3 -˛-Neu5Ac,II 3 -˛-Neu5Ac-Gg 4 Cer; GD1b, II 3 -˛-(Neu5Ac) 2 -Gg 4 Cer; GD1c, IV 3 -˛-(Neu5Ac) 2 - Gg 4 Cer; GT1b, IV 3 -˛-Neu5Ac,II 3 -˛-(Neu5Ac) 2 -Gg 4 Cer; GT1c, II 3 -˛-(Neu5Ac) 3 -Gg 4 Cer; 3’-nLM1, IV 3 -˛-Neu5Ac-nLc 4 Cer; nLD1, disialo-nLc 4 Cer. Ł Correspondence to: J. Peter-Katalini´ c, Laboratory for Biomedical Analysis, Institute for Medical Physics and Biophysics, University of M ¨ unster, Robert-Koch-Str. 31, D-48149 M ¨ unster, Germany. E-mail: jkp@uni-muenster.de † Permanent address: Laboratory for Chemistry and Biochemistry, School of Medicine, University of Zagreb, Zagreb, Croatia. of the plasma membrane, while a hydrophilic oligosaccha- ride chain protrudes into the extracellular environment. 1–3 Cells of the central nervous system (CNS), particularly neuronal membranes, contain at least several times higher concentrations of gangliosides then the extraneural cell types, highlighting their special role in the CNS. 4–6 Ganglioside species vary from each other in their carbohydrate por- tion and/or in the ceramide composition. Like all GSLs, they are classified into oligosaccharide series according to the major oligosaccharide core structure. Based only on the carbohydrate portion, more than 90 different ganglioside structures have been reported to date in neural and extra- neural tissues. 7 From 30 species found in vertebrate nervous system, half belong to the sialo-ganglio series represent- ing so-called neuronal-type structures. 8 It is reasonable to assume that these lists are not yet completed. 9 The composition of ganglioside species (pattern) expres- sed in cells is cell type-, tissue- and species-specific as well as specifically changed during development and differentiation, 10 – 12 ageing, 13 – 15 degeneration and malig- nant transformation. 16 Significant physiological implications of gangliosides, evidenced by numerous studies, can be Copyright  2001 John Wiley & Sons, Ltd.