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 proteincarbohydrate interactions. Protein-carbohydrate interactions are of profound importance in diverse biological phenomena such as extracellular matrix assembly, cell adhesion, tumor metastasis and the immune response. 1,2 When possible, high-resolution methods such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography are the preferred ways to study the three-dimensional structure of complexes between proteins and carbohydrates. However, complexes involving oligosaccharides are frequently not suitable for analysis by these techniques because they are difficult to purify in sufficient quantities and do not readily crystallize due to the hydrophilicity and dynamics of the carbohydrate component in solution. 3,4 Thus, sensitive techniques that minimize the amounts of material analyzed are needed to investigate protein-carbohydrate structure and function. One such technique, hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS), has proven...

Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

“…Notably, changes in D-uptake rates at residues remote from the binding sites have been demonstrated previously for protein-carbohydrate complexes [25,27], as well as other protein-ligand interactions [52][53][54]. Both decreases [25,52,53] and increases [25,27,53,54] in the D-uptake outside of binding sites have been observed. These data suggest that changes in the structure or dynamics of the N-terminal region Interestingly, no such ligand-induced structural changes are evident from X-ray crystallography data [38].…”

“…It can be seen in Figure 4b that the de-protected regions are mainly located close to the N-terminus, which is the artificial truncation point of the fragment [38]. Notably, changes in D-uptake rates at residues remote from the binding sites have been demonstrated previously for protein-carbohydrate complexes [25,27], as well as other protein-ligand interactions [52][53][54]. Both decreases [25,52,53] and increases [25,27,53,54] in the D-uptake outside of binding sites have been observed.…”

“…To date, however, there have been few HDX-MS studies reported for protein-carbohydrate interactions [25][26][27]. One possible reason for this is the low affinities that are typical of protein-carbohydrate interactions (association constants {K a } of~10 3 M −1 [3]).…”

“…Finally, carbohydrate binding can be accompanied by changes in protein structure and dynamics. These changes may lead to a decrease or increase in D-uptake of residues remote from the carbohydrate binding site [27,28]. Consequently, the measured differences in peptide D-uptake may reflect the formation of intermolecular interactions or ligand binding-induced changes in protein conformation or dynamics, or a combination of these effects [9,10].…”

Automated data reduction for hydrogen/deuterium exchange experiments, enabled by high-resolution fourier transform ion cyclotron resonance mass spectrometry

“…High-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) [19] provides mass resolving power (m/Δm 50% ≥ 100,000, in which Δm 50% denotes mass spectral peak full width at halfmaximum peak height) sufficient to resolve virtually all of the overlapped peptide isotopic distributions in such experiments [5-7, 10, 20-23]. In this paper, we devise an algorithm that can make those assignments reliable, rapid, and automatic, based on high-resolution, accurate Fourier transform ion cyclotron resonance mass spectral data.…”