Dataset Information

Polar aprotic modifiers for chromatographic separation and back-exchange reduction for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

ABSTRACT: Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein–protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) and supercritical fluid chromatography have been shown to decrease back-exchange. Here, we show that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent.

SUBMITTER: Valeja SG

PROVIDER: S-EPMC3835171 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Polar aprotic modifiers for chromatographic separation and back-exchange reduction for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

Valeja Santosh G SG Emmett Mark R MR Marshall Alan G AG

Journal of the American Society for Mass Spectrometry 20120401 4

Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein–protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) ...[more]

PMID: 22298288

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Project description: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.

Project description:Metabolomics is an interdisciplinary field that aims to study all metabolites < 1500 Da that are ubiquitously found within all organisms. Metabolomics is experiencing exponential growth and commonly relies on high-resolution mass spectrometry (HRMS). Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a form of HRMS that is particularly well suited for metabolomics research due to its exceptionally high resolution (105-106) and sensitivity with a mass accuracy in parts per billion (ppb). In this regard, FT-ICR-MS can provide valuable insights into the metabolomics analysis of complex biological systems due to unique capabilities such as the easy separation of isobaric and isomeric species, isotopic fine structure analysis, spatial resolution of metabolites in cells and tissues, and a high confidence (<1 ppm mass error) in metabolite identification. Alternatively, the large and complex data sets, long acquisition times, high cost, and limited access mainly through national mass spectrometry facilities may impede the routine adoption of FT-ICR-MS by metabolomics researchers. This review examines recent applications of FT-ICR-MS metabolomics in the search for clinical and non-human biomarkers; for the analysis of food, beverage, and environmental samples; and for the high-resolution imaging of tissues and other biological samples. We provide recent examples of metabolomics studies that highlight the advantages of FT-ICR-MS for the detailed and reliable characterization of the metabolome. Additionally, we offer some practical considerations for implementing FT-ICR-MS into a research program by providing a list of FT-ICR-MS facilities and by identifying different high-throughput interfaces, varieties of sample types, analysis methods (e.g., van Krevelen diagrams, Kendrick mass defect plot, etc.), and sample preparation and handling protocols used in FT-ICR-MS experiments. Overall, FT-ICR-MS holds great promise as a vital research tool for advancing metabolomics investigations.

Dataset Information

Polar aprotic modifiers for chromatographic separation and back-exchange reduction for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

Publications

Polar aprotic modifiers for chromatographic separation and back-exchange reduction for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

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