Glycosaminoglycan-Protein Binding

Summary

Principal Investigator: J Zaia
Abstract: The elucidation of the mechanisms whereby heparan sulfate (HS) binds proteins remains one of the most challenging problems in glycobiology. The binding between HS and growth factors and growth factor receptors plays an essential role in embryonic patterning and development. Many diseases have been linked to HS-protein binding events, including atherosclerosis. Nearly all mamallian cells express HS on their surfaces to mediate interactions between growth factors and growth factor receptors. Despite the central role HS plays in glycobiology, understanding of its interactions with growth factors and growth factor receptors has been severly limited by the lack of tools that are both rapid and sensitive enough to analyze it at biologically relevant levels. We are seeking to further the understanding of the HS structures that mediate growth factor binding by employing capillary equilibrium size exclusion chromatography (SEC) on-line with electrospray mass spectrometric detection. This technique will be capable of simultaneously determining the structures and binding orders of HS oligosaccharides released from cell surfaces. Equilibrium SEC carries the significant advantage that binding constants are determined purely from elution times and do not require knowledge of the ligand concentration. The use of capillary SEC columns will allow binding measurements to be made from biologically relevant levels of HS and binding proteins. Electrospray mass spectrometric detection will allow direct sequencing of the highly sulfated HS oligosaccharides using modern tandem mass spectrometric scans. The major areas proposed herein include: 1) Development and optimization of an equilibrium SEC interface for electrospray mass spectrometers; 2) Development of a scheme for identification of antithrombin Ill-binding epitopes and flanking sequences from commercial heparin and HS; 3) Determination of the expression of protein-binding sequences on particular proteoglycan gene products as a function of vascular cell type and growth conditions.
Funding Period: 2003-07-21 - 2007-06-30
more information: NIH RePORT

Top Publications

  1. pmc Effective use of mass spectrometry for glycan and glycopeptide structural analysis
    Nancy Leymarie
    Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University Medical Campus, Boston, Massachusetts 02118, USA
    Anal Chem 84:3040-8. 2012
  2. pmc Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow
    Gregory O Staples
    Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, 670 Albany Street, Boston, Massachusetts 02118, USA
    Anal Chem 82:516-22. 2010
  3. pmc Comparative glycomics using a tetraplex stable-isotope coded tag
    Michael J Bowman
    Boston University School of Medicine, Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston, Massachusetts 02118, USA
    Anal Chem 82:3023-31. 2010
  4. pmc Organ-specific heparan sulfate structural phenotypes
    Xiaofeng Shi
    Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA
    J Biol Chem 284:11806-14. 2009
  5. pmc A chip-based amide-HILIC LC/MS platform for glycosaminoglycan glycomics profiling
    Gregory O Staples
    Department of Biochemistry, Boston University School of Medicine, Mass Spectrometry Resource, Boston, MA 02118, USA
    Proteomics 9:686-95. 2009
  6. pmc Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection
    Alicia M Hitchcock
    Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
    Electrophoresis 29:4538-48. 2008
  7. pmc Mass spectrometry and the emerging field of glycomics
    Joseph Zaia
    Deptartment of Biochemistry, Boston University, 670 Albany Street, Boston, MA 02118, USA
    Chem Biol 15:881-92. 2008
  8. pmc Comparative glycomics of connective tissue glycosaminoglycans
    Alicia M Hitchcock
    Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
    Proteomics 8:1384-97. 2008
  9. pmc Characterization of heparin oligosaccharides binding specifically to antithrombin III using mass spectrometry
    Hicham Naimy
    Department of Biochemistry, Boston University School of Medicine, 670 Albany Street, Boston, Massachusetts 02118, USA
    Biochemistry 47:3155-61. 2008
  10. pmc Tags for the stable isotopic labeling of carbohydrates and quantitative analysis by mass spectrometry
    Michael J Bowman
    Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts 02118, USA
    Anal Chem 79:5777-84. 2007

Scientific Experts

  • J Zaia
  • Andre Ziegler
  • Alicia M Hitchcock
  • Michael J Bowman
  • Catherine E Costello
  • Gregory O Staples
  • Nancy Leymarie
  • Hicham Naimy
  • Xiaofeng Shi
  • Karen E Yates
  • Jennifer L Seymour
  • May Joy C Miller
  • Kevin Kileen
  • Hongfeng Yin
  • Karsten Kraiczek
  • Christine Miller
  • James M Lau
  • Sonya Shortkroff
  • Anders Malmstrom

Detail Information

Publications16

  1. pmc Effective use of mass spectrometry for glycan and glycopeptide structural analysis
    Nancy Leymarie
    Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University Medical Campus, Boston, Massachusetts 02118, USA
    Anal Chem 84:3040-8. 2012
    ..This technology enables precise characterization of recombinant glycoproteins in the pharmaceutical industry and academic biomedicine...
  2. pmc Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow
    Gregory O Staples
    Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, 670 Albany Street, Boston, Massachusetts 02118, USA
    Anal Chem 82:516-22. 2010
    ..It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest...
  3. pmc Comparative glycomics using a tetraplex stable-isotope coded tag
    Michael J Bowman
    Boston University School of Medicine, Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston, Massachusetts 02118, USA
    Anal Chem 82:3023-31. 2010
    ..The data demonstrate the value of the tetraplex stable isotope tagging approach for producing high-quality glycomics compositional profiling and fine structural analysis...
  4. pmc Organ-specific heparan sulfate structural phenotypes
    Xiaofeng Shi
    Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA
    J Biol Chem 284:11806-14. 2009
    ..The data show the presence of a disaccharide with an unsubstituted amino group that is endogenous and widely expressed in mammalian organ tissues...
  5. pmc A chip-based amide-HILIC LC/MS platform for glycosaminoglycan glycomics profiling
    Gregory O Staples
    Department of Biochemistry, Boston University School of Medicine, Mass Spectrometry Resource, Boston, MA 02118, USA
    Proteomics 9:686-95. 2009
    ..In summary, chip based amide-HILIC LC/MS is an enabling technology for GAG glycomics profiling...
  6. pmc Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection
    Alicia M Hitchcock
    Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
    Electrophoresis 29:4538-48. 2008
    ..The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue...
  7. pmc Mass spectrometry and the emerging field of glycomics
    Joseph Zaia
    Deptartment of Biochemistry, Boston University, 670 Albany Street, Boston, MA 02118, USA
    Chem Biol 15:881-92. 2008
    ..Mass spectrometry (MS) is emerging as an enabling technology in the field of glycomics. This review summarizes recent developments in mass spectrometric analysis methods for protein-based glycomics and glycoproteomics workflows...
  8. pmc Comparative glycomics of connective tissue glycosaminoglycans
    Alicia M Hitchcock
    Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
    Proteomics 8:1384-97. 2008
    ..The results provide important new information on the changes to the expression of CS GAG chains during disease and development...
  9. pmc Characterization of heparin oligosaccharides binding specifically to antithrombin III using mass spectrometry
    Hicham Naimy
    Department of Biochemistry, Boston University School of Medicine, 670 Albany Street, Boston, Massachusetts 02118, USA
    Biochemistry 47:3155-61. 2008
    ..The specificity of the hexasaccharides binding ATIII was confirmed by assaying their ability to enhance ATIII-mediated inhibition of Factor Xa in vitro...
  10. pmc Tags for the stable isotopic labeling of carbohydrates and quantitative analysis by mass spectrometry
    Michael J Bowman
    Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts 02118, USA
    Anal Chem 79:5777-84. 2007
    ..Quantitation of partially depolymerized glycosaminoglycan mixtures, as well as N-linked glycans released from fetuin, is used to demonstrate the utility of the tetraplex tagging strategy...
  11. pmc The role of mobile protons in negative ion CID of oligosaccharides
    Joseph Zaia
    Department of Biochemistry, Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118, USA
    J Am Soc Mass Spectrom 18:952-60. 2007
    ..Mechanisms for product ion formation are proposed based on tandem mass spectra and the abundances of product ions as a function of collision energy...
  12. pmc Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling
    Alicia M Hitchcock
    Department of Biochemistry, Boston University School of Medicine, MS Resource, 670 Albany Street, Boston, MA 02118, USA
    Glycobiology 17:25-35. 2007
    ..These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue...
  13. ncbi Size-exclusion chromatography of heparin oligosaccharides at high and low pressure
    Andre Ziegler
    Proteoglycan and Glycosaminoglycan Structure Laboratory, Mass Spectrometry Resource, Boston University, 715 Albany Street, Boston, MA 02118, USA
    J Chromatogr B Analyt Technol Biomed Life Sci 837:76-86. 2006
    ..Optimized SEC affords separation of characteristic heparin trisaccharides that contain uronic acid at the reducing end and suggest cellular storage of heparin as a free glycan...
  14. pmc The influence of sialylation on glycan negative ion dissociation and energetics
    Jennifer L Seymour
    Department of Biochemistry, Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118, USA
    J Am Soc Mass Spectrom 17:844-54. 2006
    ..These results have strong practical implications because a major portion of glycans released from mammalian proteins will be sialylated...
  15. pmc A tandem mass spectrometric approach to determination of chondroitin/dermatan sulfate oligosaccharide glycoforms
    May Joy C Miller
    Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, R 806, Boston, MA 02118, USA
    Glycobiology 16:502-13. 2006
    ..Decorin samples ranged from 30% CSB-like repeats for those samples from articular cartilage to 75% for those from sclera. These values agree with known levels of glucuronyl C5-epimerase in these tissues...
  16. pmc Glycoform quantification of chondroitin/dermatan sulfate using a liquid chromatography-tandem mass spectrometry platform
    Alicia M Hitchcock
    Department of Biochemistry, Boston University School of Medicine, 670 Albany Street, Boston, Massachusetts 02118, USA
    Biochemistry 45:2350-61. 2006
    ..This work presents the first application of a glycomics platform for the quantification of CS oligosaccharide mixtures for obtaining specific information about the positions of GalNAc sulfation and uronic acid epimerization...