title: Tandem mass spectra of glycan substructures enable the multistage mass spectrometric identification of determinants on oligosaccharides.
abstract: BACKGROUND
  Glycosylation of proteins and lipids affects many biological processes, such as host-pathogen interactions, cell communication, and initiation of the immune responses. Terminal glycan substructures, or determinants, often govern the function or recognition of the carrier glycoconjugate and modulate these processes. In this study we describe a strategy using multistage mass spectrometry to identify and confirm these glycan substructures.
  
  
  METHODS
  An online tandem mass spectrometry (MS(2)) spectral fragment library of glycan substructures that typically occur at the non-reducing terminus of glycoconjugates was created to enable the easier identification and confirmation of glycan determinants on oligosaccharides released from glycoproteins. Oligosaccharides were separated by porous graphitized carbon capillary chromatography and analysed by ion trap MS. Candidate product ions that constitute the glycan substructure mass were identified in the MS(2) product ion spectrum, and used as the precursor ion for subsequent MS(3) fragmentation. The resulting MS(3) spectrum was matched against the MS(2) spectral fragment library to identify the glycan substructure(s) that comprise the parent oligosaccharide.
  
  
  RESULTS
  Thirty biologically important terminal glycan determinants commonly observed on glycoconjugates were fragmented by positive and negative ion mass spectrometry and the MS(2) product ion masses manually annotated and stored in the UniCarb-DB online database. Negative ion tandem mass spectra were especially useful in assigning isobaric glycan structures. We have applied this strategy for the identification of the sulphation, blood group antigens and sialic acid linkages on complex N-and O-glycans released from glycoproteins.
  
  
  CONCLUSIONS
  We show the potential of these glycan substructure MS(2) spectra in the negative ionization mode to facilitate the assignment of determinants on N- and O-linked glycans released from glycoproteins. Comparing the structural feature ions of known glycan reference substructures assists in the annotation of complex glycan product ion spectra, and can remove the need for other orthogonal confirmation analyses such as sequential glycosidase digestion.
authors.affiliation: Biomolecular Frontiers Research Centre, Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
authors.forename: Arun V
authors.initials: AV
authors.lastname: Everest-Dass
authors.forename: Daniel
authors.initials: D
authors.lastname: Kolarich
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Campbell
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
mesh_terms: D000017:ABO Blood-Group System / Q000032:analysis; D002240:Carbohydrate Sequence; D006001:Glycoconjugates / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D009844:Oligosaccharides / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000017:ABO Blood-Group System; D006001:Glycoconjugates; D006023:Glycoproteins; D009844:Oligosaccharides; D011134:Polysaccharides; D019158:N-Acetylneuraminic Acid