| This dissertation explores methodologies for determining the fine structure of glycosaminoglycans (GAGs), and specifically those sequences of biologically relevant heparin/HS oligosaccharides. Mass spectrometric (MS) approaches for the structural characterization of glycosaminoglycans were developed, with two main parts to the endeavor. First, a combination of methods development and fundamental studies was used for the structural analysis of glycosaminoglycan disaccharides and their isomers. Second, this dissertation also addresses the application of these new methodologies to some genuine biologically relevant questions.;A method was first developed utilizing a combination of electrospray ionization (ESI)-MS and tandem MS (MSn) for the compositional analysis of disaccharide constituents of heparin/HS upon enzymatic depolymerization. Utilizing collision-induced dissociation (CID), in a quadrupole ion trap mass spectrometer, isomeric heparin disaccharides could be differentiated and quantified. Fundamental isotopic labeling studies were undertaken using 18O and 2H to study the dissociation mechanisms of these saccharides upon CID. These MS n data also provided a foundation for further use in a practical MS-based analysis tool for the structural elucidation of larger, biologically important heparin/HS oligosaccharides.;As applications, the compositional profiling of heparin was used to assay for the heparin substrate specificity of a novel human endosulfatase, implicated in tumorigenesis. Analysis of two additional GAGs was also undertaken to determine contaminating hyaluronan content in chondroitin sulfate samples prepared for use in nutritional supplements. Finally, a complete, integrated methodology was presented utilizing a combination of enzymatic digestion, ESI-MS and MS n for the sequencing of small heparin oligosaccharides. A basic software application, or Heparin Oligosaccharide Sequencing Tool (HOST), was developed to aid in the integration and analysis of the data generated from these experiments, facilitating the process to arrive at sequence information.;MSn provides a very rapid and efficient tool for oligosaccharide analysis when limited amounts of material are available, and with HOST facilitating the interpretation of the MSn spectra obtained, this provides a practical methodology for the future analysis of heparin/HS oligosaccharides of unknown structure. To demonstrate the feasibility of this methodology, these developments were used to obtain sequence information of various heparin oligosaccharides, and furthermore, their potential for use in identifying additional sequence-specific glycosaminoglycan-protein interactions was illustrated. |
