Studying protein‐carbohydrate interactions by amide hydrogen/deuterium exchange mass spectrometry

King, Daniel; Lumpkin, Michelle; Bergmann, Carl; Orlando, Ron

doi:10.1002/rcm.746

Cited by 14 publications

(11 citation statements)

References 24 publications

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“…Intermolecular H-bonds between the ligand and backbone amides generally lead to a decrease in Duptake. Notably, this observation is consistent with those reported previously for other protein-carbohydrate complexes [26]. That protection is also conferred by water-mediated Hbonds involving backbone amides, has not, to our knowledge, been previously reported for protein-carbohydrate interactions, although it was demonstrated for other noncovalent protein complexes [15,50].…”

Section: Influence Of Protein-carbohydrate Interactions On Deuterium supporting

confidence: 93%

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

Zhang

Kitova

et al. 2015

J. Am. Soc. Mass Spectrom.

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Abstract. The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM 1 pentasaccharides (β-Gal-, respectively, served as model systems for this study.Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

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Section: Influence Of Protein-carbohydrate Interactions On Deuterium supporting

confidence: 93%

“…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]).…”

mentioning

confidence: 99%

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

Zhang

Kitova

et al. 2015

J. Am. Soc. Mass Spectrom.

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“…To obtain site-specific exchange information, the deuterated protein is rapidly digested after quenching with an acid-stable protease, such as pepsin, and analyzed by electrospray ionization (ESI) or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. 14,15 The H/D-EX MS approach can also be used to probe proteinligand interactions. In this case, the difference in deuterium uptake between the free and bound complexes could, in principle, reveal certain solvent-accessible amides involved in 18,19 In the present study ESI Fourier transform mass spectrometry (FTMS) was used to monitor an interaction involving human tumor necrosis factor stimulated gene-6 (TSG-6) and hyaluronan oligosaccharides. Hyaluronan (HA) is a linear glycosaminoglycan comprised entirely of repeating disaccharide units of…”

mentioning

confidence: 99%

“…18,19 In the present study ESI Fourier transform mass spectrometry (FTMS) was used to monitor an interaction involving human tumor necrosis factor stimulated gene-6 (TSG-6) and hyaluronan oligosaccharides. Hyaluronan (HA) is a linear glycosaminoglycan comprised entirely of repeating disaccharide units of β(1 → 3)-Nacetyl-D-glucosamine-β(1 → 4)-D-glucuronic acid.…”

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confidence: 99%

Fourier transform mass spectrometry to monitor hyaluronan–protein interactions: use of hydrogen/deuterium amide exchange

Seyfried¹,

Atwood²,

Yongye³

et al. 2006

Rapid Comm Mass Spectrometry

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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...

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“…H/D exchange MS was applied to the protein-carbohydrate complex endopolygalacturonase II (EPG-II) from Aspergillus niger and an octamer of galacturonic acid (26). The location of the binding cleft on the protein surface could be deduced by monitoring the differences in deuterium incorporation of EPG-II in the presence and absence of the carbohydrate.…”

Section: Other Methodsmentioning

confidence: 99%

Lectins: Proteins That Interpret the Sugar Code

Nilsson¹

2003

Anal. Chem.

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Studying protein‐carbohydrate interactions by amide hydrogen/deuterium exchange mass spectrometry

Cited by 14 publications

References 24 publications

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

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

Fourier transform mass spectrometry to monitor hyaluronan–protein interactions: use of hydrogen/deuterium amide exchange

Lectins: Proteins That Interpret the Sugar Code

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