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Joshua S. Sharp
Associate Research Scientist

Mass spectrometry for characterizing the structure of proteins, carbohydrates, and protein-carbohydrate complexes

Telephone: 706-542-3712
Fax: 706-542-4412

Short Biography
Research Interests
Lab-personal web site

Course BCMB4110

Short Biography:
Dr. Sharp received his B.S. in Microbiology in 1999 from the University of Tennessee in Knoxville, and his Ph.D. in Genome Science and Technology in 2003 under the direction of Dr. Robert Hettich of the Chemical Sciences Division at Oak Ridge National Laboratory. Dr. Sharp performed his post-doctoral research at the National Institute of Environmental Health Sciences (NIH) in the laboratory of Dr. Kenneth Tomer, working on the development and application of protein chemistry coupled to mass spectrometry for probing the biophysical properties of large protein complexes of biomedical interest. Dr. Sharp joined the CCRC in 2007.

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Research Interests:
Research in the Sharp laboratory focuses on the combination of biological chemistry and mass spectrometry to address problems in protein structural characterization, protein modification by oxidative stress, and detailed structural analysis of sulfated glycosaminoglycans.

Hydroxyl Radical Protein Footprinting

Our efforts in protein structural characterization have focused mainly on coupling mass spectrometry with protein-hydroxyl radical chemistry in a technique known as hydroxyl radical protein footprinting. Hydroxyl radicals are small, polar, highly reactive species that can modify many amino acid side chains in a single experiment. When produced in solution with a protein, the radicals will modify amino acid side chains on the surface of the folded protein. The rate of modification of a particular side chain is a function of its inherent chemical reactivity and its solvent accessibility. So we can compare protein samples in two conformations (e.g. carbohydrate-bound versus carbohydrate-free, dimer vs. tetramer, etc.) by reacting the protein in each state with hydroxyl radicals and then analyzing the amount of oxidation at each oxidation site. Reduced amounts of oxidation that occurs in one of the two states indicate a change in the structure that shielded those particular sites from the hydroxyl radical. By measuring these changes in solvent accessibility for many amino acid residues in the protein, we can use these data to help generate structural models that reflect the changes in shape in the protein. We have applied this technology on various carbohydrate-binding proteins and protein complexes, with the goal of characterizing the carbohydrate binding surface(s) as well as carbohydrate binding-induced conformational changes. We are currently using these techniques to study protein-glycosaminoglycan interactions in protein chemokine systems important in immune response, as well as proteins involved in pathogen-target cell interactions and HIV-neutralizing antibodies.

As one of the early pioneers in this area of research, the Sharp group is also active in developing improvements in both the protein chemistry and the analytical tools used for hydroxyl radical footprinting, in order to improve the spatial resolution, robustness, and ease of use of the technology, as well as extending applications to classes of systems currently unapproachable by the technique. Recent advances in hydroxyl radical sources have allowed us to generate hydroxyl radicals in sub-microsecond bursts, achieving heavy oxidation of the protein on a timescale faster than the protein can unfold due to the oxidative modifications. Similarly, we have also developed techniques for generating these rapid bursts of radicals in the absence of reactive precursor oxidants, generating hydroxyl radicals directly from water using a Van de Graaff electron accelerator. Current work includes improved fragmentation techniques for quantitative MS/MS of oxidized peptides and proteins, improved separations techniques using both UPLC and ion mobility to better separate oxidized from unoxidized peptides, and improved semi-automated analysis methods for mining MS/MS data for hydroxyl radical footprinting purposes.

Protein Modification in Oxidative Stress

Oxidative stress is an important factor in a wide variety of disease processes, both as a factor in pathology in diseases and disorders such as Alzheimer’s disease, and as a part of the immunological response against pathogens. The reactive oxygen species (ROS) present in conditions of oxidative stress react with a wide variety of biomolecules; however, the majority of ROS-mediated damage is thought to occur on proteins. Oxidative damage of proteins are known to alter their structure, dynamics, and function in a very complex manner. One of the areas of interest in the Sharp group is the development and application of techniques to study the effects of ROS on various proteins. We focus on the development and application of techniques using chromatography and mass spectrometry for identifying and quantifying sites of oxidative damage in proteins and peptides. As part of these efforts, we have developed a method for dose-dependent hydroxyl radical protein footprinting. Using this method, we can study changes in the structure of proteins that occurs due to oxidative damage. We have utilized this method to study structural changes in signaling proteins known to be sensitive to oxidation. Additionally, we also develop and use pseudo-MRM based methods for quantifying protein oxidation and repair at multiple sites simultaneously to aid our collaborators in studying the damage and repair of various proteins sensitive to oxidative stress within both human cells and pathogens.

Structural Characterization of Sulfated Glycosaminoglycans

Glycosaminoglycans (GAGs) are biologically important linear polysaccharides, many of which are characterized by a complex pattern of N- and/or O-sulfation. These sulfation patterns have been shown to be crucial for GAG protein recognition and function. However, while analysis of GAG disaccharides is now routine, sequencing of these sulfation patterns within larger oligomers has presented tremendous difficulties. The sulfation patterns are complex, and standard LC methods are often insufficient to separate isomeric sequences. Additionally, loss of the sulfate group is common in standard CID MS/MS, which results in loss of information regarding the original site of sulfation. While considerable progress has been made in electron based fragmentation of these oligomers, this technique is currently unable to handle mixtures of isomeric GAGs.

The Sharp group has developed a technique for sequencing these GAGs using a mixture of derivatization chemistry and LC-MSn analysis. Briefly, unsulfated regions are protected by permethylation chemistry. Sulfations are then removed by solvolysis, and the regions exposed by desulfations are labeled with a peracetylation reaction. The derivatized GAG is amenable to high-resolution reverse phase chromatography, which is quite capable of separating sulfation isomers in an electrospray-friendly solvent system. When coupled with mass spectrometry, we are able to identify sites of GAG modification (including sulfation) through controlled CID fragmentation. We have successfully structurally characterized pure oligosaccharides up to dodecasaccharides, as well as separated and characterized an isomeric mixture of hexasaccharides. Current work is focused on identification of uncommon sulfation sites, improvements in sensitivity, alternative derivatization chemistries to improve analytical performance, and incorporation of ion mobility separations into a multidimensional separation/mass spectrometry method.

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Publications: Author's Last Name: Sharp

Journal Articles
Book Chapters are listed at the bottom of this page.

R Huang, C Zong, A Venot, Y Chiu, D Zhou, GJ Boons, JS Sharp. 2016. De novo sequencing of complex mixtures of heparan sulfate oligosaccharides. Anal Chem 88(10): 5299-5307. PMID:27087275

C. Zong, R. Huang, E. Condac, Y Chiu, W.Y. Xiao, X. Li, W Lu, M Ishahara, S. Wang, A. Ramiah, M Stickney, P. Azadi, IJ Amster, KW Moremen, L. Wang, JS Sharp, GJ Boons. 2016. Integrated approach to identify heparan sulfate ligand requirements of Robo1. J Am Chem Soc 138(39): 13059- 13067. PMID:27611601

Z. Liz, HA Moniz, A. Ramiah, KW Moremen, JS Sharp. 2015. High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo-1-Heparin Binding Interface. J. Biol. Chem. In press.

R. Huang, Z.-J. Liu, J.S. Sharp. 2013. An approach for separation and complete structural sequencing of heparin/heparan sulfate-like oligosaccharides. Anal. Chem. 85: 5787-5795. PMID:23659663

X. Li, Z. Li, B. Xie, J.S. Sharp. 2013. Improved identification and relative quantification of sites of peptide and protein oxidation for hydroxyl radical footprinting . J. Am. Soc. Mass Spectrom. 24: 1767-1776. PMID:24014150

L.G. Kuhns, M. Mahawar, J.S. Sharp, S. Benoit, R.J. Maier. 2013. Role of Heliobacter pylori methionine sulfoxide reductase in urease maturation. Biochem. J. 450: 141-148. PMID:23181726

X Wang, JS Sharp, TM Handel, JH Prestegard. 2013. Chemokine oligomerization in cell signaling and migration. Prog. Mol. Biol. Transl. Sci. 117: 531-578. PMID:23663982

S.L. Benoit, K. Bayyareddy, M. Mahawar, J.S. Sharp, R.J. Maier. 2013. Heliobacter pylori peptide methionine sulfoxide reductase repairs critical methionine residues in alkyl hydroperoxide reductase . J. Bacteriol. In press.: -.

V.H. Pomin, Y. Park, R. Huang, C. Heiss, H,H, Sharp, P. Azadi, J.H. Prestegard. 2012. Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: production of well-defined hexasaccharides. Glycobiol. 22: 826-838. PMID:22345629

M. Mahawar, V. Tran, J.S. Sharp, R.J. Baier. 2011. Synergistic roles of Heliobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant damaged catalase. . J. Biol. Chem. 286: 19159-19169. PMID:21460217

V.H. Pomin, J.S. Sharp, X. Li, L. Wang, J.H. Prestegard. 2010. Characterization of glycosaminoglycans by 15N NMR spectroscopy and in vivo isotopic labeling. Anal. Chem. 82: 4078-4088. PMID:20423049

M. Bern, J. Saladino, J.S. Sharp. 2010. Conversion of methionine into homocysteic acid in heavily oxidized proteomics samples. Rapid Commun. Mass Spectrom. 24: 768-772. PMID:20169556

J. Saladino, M.T. Swulius, D. Live, J.S. Sharp. 2009. Alipahatic peptidyl hydroperoxides as a source of secondary oxidation in hydroxyl radical protein footprinting. J. Amer. Soc. Mass Spectrom. 20: 1123-1126. PMID:19278868

C. Watson, I. Janik, T. Zhuang, O. Charvatova, R.J. Woods, J.S. Sharp. 2009. Pulsed electron beam water radiolysis for submicrosecond hydroxyl radical protein footprinting. Anal. Chem. 81: 2496-2505. PMID:19265387

B.C. Gau, J.S. Sharp, D.L. Rempel, M.L. Gross. 2009. Fast photochemical oxidation of proteins footprints faster than protein unfolding. Anal. Chem. 81: 6563-6571. PMID:20337372

O. Charvatova, B.L. Foley, M. Bern, J. Sharp, R. Orlando, R.J. Woods. 2008. Quantifying protein interface footprinting by hydroxyl radical oxidation and molecular dynamics simulation: application to galectin-1. J. Am. Soc. Mass Spectrom. 19: 1692-1705. PMID:18707901

J.G. Smedley, J.S. Sharp, J.F. Kuhn, K.B. Tomer. 2008. Probing the pH-dependent prepore to pore transition of Bacillus anthracis protective antigen with differential oxidative protein footprinting. Biochemistry 47: 10694-10704. PMID:18785752

Book Chapters

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