title: Fourier transform mass spectrometry to monitor hyaluronan-protein interactions: use of hydrogen/deuterium amide exchange.
abstract: The use of Fourier transform mass spectrometry (FTMS) to monitor noncovalent complex formation in the gas phase under native conditions between the Link module from human tumor necrosis factor stimulated gene-6 (Link_TSG6) and hyaluronan (HA) oligosaccharides is reported. In particular, a titration experiment with increasing concentrations of octasaccharide (HA(8)) to protein produced a noncovalent complex with 1:1 stoichiometry when the oligosaccharide was in molar excess. However, in the presence of a molar excess of tetrasaccharide (HA(4)) nearly all proteins and oligosaccharides were observed in their unbound charge states. These results are consistent with solution-phase properties for this interaction in which HA(8), but not HA(4), supports high affinity Link_TSG6 binding. Hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS) was also utilized to investigate the level of global deuterium incorporation, over time, for Link_TSG6 in both the absence and presence of HA(8). After dilution into quenching conditions, deuterium incorporation reached limiting asymptotic values of 37 and 26 deuterons for the free and bound protein at 240 and 480 min, respectively, indicating that the oligosaccharide interferes with amide exchange on binding. To detect sequence-specific deuterium incorporation, pepsin digestion of Link_TSG6 in both the absence and presence of HA(8) was performed. A level of deuterium incorporation of 10-30% was observed for peptides analyzed in free Link_TSG6. Interestingly, HA(8) blocked some sites of proteolysis in Link_TSG6 compared to the free protein. Molecular modeling indicated that amino acids proximal to the ligand correlated with regions of the protein that were resistant to enzymatic digestion. Of the peptides that could be analyzed by H/D-EX MS in the presence of the ligand, a 30-60% reduction in deuterium incorporation, relative to the free protein, was observed, even for those sequences not directly involved in HA binding. These results support the utility of FTMS as a method for the characterization of protein-carbohydrate interactions.
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Seyfried
authors.forename: James A
authors.initials: JA
authors.lastname: Atwood
authors.forename: Austin
authors.initials: A
authors.lastname: Yongye
authors.forename: Andrew
authors.initials: A
authors.lastname: Almond
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
authors.forename: Ron
authors.initials: R
authors.lastname: Orlando
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Woods
mesh_terms: D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism*; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D011485:Protein Binding; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
chemical_list: D015815:Cell Adhesion Molecules; C072273:TNFAIP6 protein, human; D006820:Hyaluronic Acid; D003903:Deuterium