title: Mass spectrometric analysis of glycosphingolipid antigens.
abstract: Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition(1-2). They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Galα4Galβ4Glcβ1Cer) and isoglobotriaosylceramide (Galα3Galβ4Glcβ1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions(3-4). In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases(5). The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions(6). Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells(7-9). This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids(10-15), followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present(9). Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.
authors: Undergraduate Program, Rice University, USA.
authors: Alexandra Bili
authors: AB
authors: Yin
authors: David
authors: D
authors: Hawke
authors: Dapeng
authors: D
authors: Zhou
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D006028:Glycosphingolipids / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D006028:Glycosphingolipids
references: J Am Chem Soc. 2003 Dec 31;125(52):16213-9
references: 14692762
references: Anal Chem. 2005 Oct 1;77(19):6250-62
references: 16194086
references: J Biol Chem. 1994 Oct 28;269(43):27108-14
references: 7929454
references: J Biol Chem. 1984 Apr 25;259(8):4792-801
references: 6715324
references: J Biochem. 1964 Feb;55:205-8
references: 14135466
references: Anal Chem. 2005 Oct 1;77(19):6263-70
references: 16194087
references: Glycobiology. 2008 Feb;18(2):158-65
references: 18056651
references: J Exp Med. 1986 Jun 1;163(6):1391-404
references: 3519828
references: Nature. 2001 Oct 4;413(6855):531-4
references: 11586362
references: Glycobiology. 2008 Feb;18(2):166-76
references: 18048405
references: J Proteome Res. 2009 Jun;8(6):2740-51
references: 19284783
references: Front Biosci (Schol Ed). 2011 Jun 01;3:1520-32
references: 21622287
references: Rapid Commun Mass Spectrom. 2005;19(23):3421-8
references: 16252310
references: Methods Enzymol. 1987;138:13-25
references: 3600318
references: Curr Protein Pept Sci. 2006 Aug;7(4):325-33
references: 16918447