title: LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization.
abstract: The biosynthesis of glycans is a template-free process; hence compositionally identical glycans may contain highly heterogeneous structures. Meanwhile, the functions of glycans in biological processes are significantly influenced by the glycan structure. Structural elucidation of glycans is an essential component of glycobiology. Although NMR is considered the most powerful approach for structural glycan studies, it suffers from low sensitivity and requires highly purified glycans. Although mass spectrometry (MS)-based methods have been applied in numerous glycan structure studies, there are challenges in preserving glycan structure during ionization. Permethylation is an efficient derivatization method that improves glycan structural stability. In this report, permethylated glycans are isomerically separated; thus facilitating structural analysis of a mixture of glycans by LC-MS/MS. Separation by porous graphitic carbon liquid chromatography at high temperatures in conjunction with tandem mass spectrometry (PGC-LC-MS/MS) was utilized for unequivocal characterization of glycan isomers. Glycan fucosylation sites were confidently determined by eliminating fucose rearrangement and assignment of diagnostic ions, achieved by permethylation and PGC-LC at high temperatures, respectively. Assigning monosaccharide residues to specific glycan antennae was also achieved. Galactose linkages were also distinguished from each other by CID/HCD tandem MS. This was attainable because of the different bond energies associated with monosaccharide linkages. Graphical Abstract LC-MS and tandem MS of terminal galactose isomers.
authors: Department of Chemistry and Biochemistry, Texas Tech University, Memorial Circle & Boston, Box 41061, Lubbock, TX, 79409-1061, USA.
authors: Shiyue
authors: S
authors: Zhou
authors: Department of Chemistry and Biochemistry, Texas Tech University, Memorial Circle & Boston, Box 41061, Lubbock, TX, 79409-1061, USA.
authors: Xue
authors: X
authors: Dong
authors: Department of Chemistry and Biochemistry, Texas Tech University, Memorial Circle & Boston, Box 41061, Lubbock, TX, 79409-1061, USA.
authors: Lucas
authors: L
authors: Veillon
authors: Department of Chemistry and Biochemistry, Texas Tech University, Memorial Circle & Boston, Box 41061, Lubbock, TX, 79409-1061, USA.
authors: Yifan
authors: Y
authors: Huang
authors: Department of Chemistry and Biochemistry, Texas Tech University, Memorial Circle & Boston, Box 41061, Lubbock, TX, 79409-1061, USA. yehia.mechref@ttu.edu.
authors: Yehia
authors: Y
authors: Mechref
mesh_terms: D002853:Chromatography, Liquid; D007536:Isomerism; D008745:Methylation; D011134:Polysaccharides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D011134:Polysaccharides
keywords: LC-MS/MS; N-Glycan isomers; Permethylation; Porous graphitic carbon column; Tandem mass spectrometry