Improving digestion efficiency under H/D exchange conditions with activated pepsinogen coupled columns

The ability to analyze localized amide hydrogen/deuterium (H/D) exchange kinetics of a protein is highly dependent on the digestion efficiency of the enzyme being used to generate a collection of labeled peptides. Pepsin is one of the most commonly used acid proteases in amide H/D exchange experiments due to its broad specificity and robustness at “slow exchange” conditions of low temperature and pH. The chemistry used to immobilize pepsin to POROS AL-20 beads is optimal under neutral pH conditions but acid proteases such as pepsin are irreversibly denatured above pH 5 so coupling must be performed under suboptimal conditions. Thus, in the current study we report a technique to improve the digestion efficiency for amide H/D exchange by immobilizing pepsinogen to POROS AL-20 support under optimal coupling conditions of pH 6.7 where pepsinogen is stable and subsequently converting the coupled pepsinogen into active pepsin. This activated pepsinogen column demonstrated a higher specific activity at both 25 °C and 0 °C than an identically coupled pepsin column and gave better peptide coverage for cytochrome C and manganese superoxide dismutase (MnSOD) improving our amide H/D exchange data for these proteins. These results were reproducible for three independently coupled and activated pepsinogen columns. Protein assays demonstrated that more enzyme was bound to the POROS AL-20 resin coupled with pepsinogen at pH 6.7 than pepsin at pH 5.     

Differentiation of sulfate and phosphate by H/D exchange mass spectrometry: application to isoflavone

Often phosphorylation or sulfation is an important step which occurs in the signal transduction and cascade of metabolic pathways. Some natural products and metabolites contain one or more sulfate or phosphate groups. Isoflavone sulfate has been identified from high-resolution mass spectrometry (HRMS) and enzymatic digestion by sulfatase. We previously reported the new water-soluble isoflavone analogs, daidzein 7-O-phosphate and genistein 7-O-phosphate, which were surprisingly hydrolyzed by sulfatase. In this previous study, we could not determine the phosphate from the results of HRMS and enzymatic digestion, that is, HRMS and enzymatic digestion did not provide clear evidence. In this case, we drew conclusions from NMR analysis. HRMS has been ineffective with a regular fast atom bombardment (FAB) mass spectrometer to distinguish between phosphate and sulfate since the mass difference is only 0.009 mass units. There was, however, no conventional method of microanalysis to distinguish phosphate from sulfate owing to the same nominal mass. It is still very difficult to determine by negative FABMS [--O--P(==O)(OH)(2)] = 80 and [--O--S(==O)(2)OH] = 80. In this paper, we report a method to distinguish between these groups by using a popular low-resolution mass instrument; thus, phosphate and sulfate were measured by H/D exchange mass spectrometry at the picomole level to differentiate [--O--P(==O)(OD)(2)] = 82 and [--O--S(==O)(2)OD] = 81, respectively. This method is applicable not only to the isoflavone, but also to other phospho and sulfo compounds.     

An efficient and inexpensive refrigerated LC system for H/D exchange mass spectrometry

Loss of deuterium label during the LC step in amide hydrogen/deuterium exchange mass spectrometry (H/D-MS) is minimized by maintaining an acidic mobile phase pH and low temperature (pH 2.5, 0 °C). Here we detail the construction and performance of a low-cost, thermoelectrically refrigerated enclosure to house high-performance liquid chromatography (HPLC) components and cool mobile phases. Small volume heat exchangers rapidly decrease mobile phase temperature and keep the temperature stable to ±0.2 °C. Using a superficially porous reversed-phase column, we obtained excellent chromatographic performance in the separation of peptides with a median peak width of 4.4 s. Average deuterium recovery was 80.2% with an average relative precision of 0.91%.     

Quantification of protein-ligand interactions by mass spectrometry,titration, and H/D exchange: PLIMSTEX

Protein-ligand binding and the concomitant conformational change in the protein are of crucial importance in biophysics and drug design. We report a novel method to quantify protein-ligand interactions in solution by mass spectrometry, titration, and H/D exchange (PLIMSTEX). The approach can determine the conformational change, binding stoichiometry, and affinity in protein-ligand interactions including those that involve small molecules, metal ions, and peptides. Binding constants obtained by PLIMSTEX for four model protein-ligand systems agree with K values measured by conventional methods. At higher protein concentration, the method can be used to determine quickly the binding stoichiometry and possibly the purity of proteins. Taking advantage of concentrating the protein on-column and desalting, we are able to use different concentrations of proteins, buffer systems, salts, and pH in the exchange protocol. High picomole quantities of proteins are sufficient, offering significantly better sensitivity than that of NMR and X-ray crystallography. Automation could make PLIMSTEX a high throughput method for library screening, drug discovery, and proteomics.     

Fast reversed-phase liquid chromatography to reduce back exchange and increase throughput in H/D exchange monitored by FT-ICR mass spectrometry

In solution-phase hydrogen/deuterium exchange (HDX), it is essential to minimize the back-exchange level of H for D after the exchange has been quenched, to accurately assign protein conformation and protein-protein or protein-ligand interactions. Reversed-phase HPLC is conducted at low pH and low temperature to desalt and separate proteolytic fragments. However, back exchange averages roughly 30% because of the long exposure to H₂O in the mobile phase. In this report, we first show that there is no significant backbone amide hydrogen back exchange during quench and digestion; backbone exchange occurs primarily during subsequent liquid chromatography separation. We then show that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, resulting from the dramatically reduced retention time of the peptide fragments on the column. The influence of retention time on back exchange was also evaluated. The rapid separation coupled with high-resolution FT-ICR MS at 14.5 T provides high amino acid sequence coverage, high sample throughput, and high reproducibility and reliability.     

Modeling data from titration,amide H/D exchange,and mass spectrometry to obtain protein-ligand binding constants

We recently reported a new method for quantification of protein-ligand interaction by mass spectrometry, titration and H/D exchange (PLIMSTEX) for determining the binding stoichiometry and affinity of a wide range of protein-ligand interactions. Here we describe the method for analyzing the PLIMSTEX titration curves and evaluate the effect of various models on the precision and accuracy for determining binding constants using H/D exchange and a titration. The titration data were fitted using a 1:n protein:ligand sequential binding model, where n is the number of binding sites for the same ligand. An ordinary differential equation was used for the first time in calculating the free ligand concentration from the total ligand concentration. A nonlinear least squares regression method was applied to minimize the error between the calculated and the experimentally measured deuterium shift by varying the unknown parameters. A resampling method and second-order statistics were used to evaluate the uncertainties of the fitting parameters. The interaction of intestinal fatty-acid-binding protein (IFABP) with a fatty-acid carboxylate and that of calmodulin with Ca(2+) are used as two tests. The modeling process described here not only is a new tool for analyzing H/D exchange data acquired by ESI-MS, but also possesses novel aspects in modeling experimental titration data to determine the affinity of ligand binding.     

Accuracy of SUPREX (stability of unpurified proteins from rates of H/D exchange) and MALDI mass spectrometry-derived protein unfolding free energies determined under non-EX2 exchange conditions

Described here is the impact of so-called non-EX2 exchange behavior on the accuracy of protein unfolding free energies (i.e., DeltaG u values) and m values (i.e.,-deltaDeltaG u/delta[denaturant] values) determined by an H/D exchange and mass spectrometry-based technique termed stability of unpurified proteins from rates of H/D exchange (SUPREX). Both experimental and theoretical results on a model protein, ubiquitin, reveal that reasonably accurate thermodynamic parameters for its folding reaction can be determined by SUPREX even when H/D exchange data is collected in a non-EX2 regime. Not surprisingly, the theoretical results reported here on a series of hypothetical protein systems with a wide range of biophysical properties show that the accuracy of SUPREX-derived DeltaG u and m values is compromised for many proteins when analyses are performed at high pH (e.g., pH 9) and for selected proteins with specific biophysical parameters (e.g., slow folding rates) when analyses are performed at lower pH. Of more significance is that the experimental and theoretical results reveal a means by which problems with non-EX2 exchange behavior can be detected in the SUPREX experiment without prior knowledge of the protein's biophysical properties. The results of this work also reveal that such problems with non-EX2 exchange behavior can generally be minimized if appropriate H/D exchange times are employed in the SUPREX experiment to yield SUPREX curve transition midpoints at chemical denaturant concentrations less than 2 M.     

Capillary zone electrophoresis of glycopeptides under controlled electroosmotic flow conditions coupled to electrospray and matrix-assisted laser desorption/ionization mass spectrometry

Defined conditions of EOF along with different pH values of the BGE were compared for the purpose of analyzing glycopetides by CZE coupled to MS (CZE-MS). Hyphenation to MS involved ESI as well as MALDI, and single-stage and multistage MS were applied. Variation of the EOF was accomplished by selecting appropriate coatings for the capillary, namely hexadimethrine bromide (HDMB) and HDMB/dextran sulfate. A high and reproducible anodic and cathodic EOF, respectively, was obtained in both approaches, allowing the detection of analytes with net positive as well as negative charge in one single run. Thus, a fast and sensitive determination of the glycopeptides in a tryptic digest of antithrombin, chosen as a test sample, was achieved. Ionization suppression effects, a phenomenon typically observed with glycopeptides in MS analysis, were minimized thanks to separation from other peptides present. The high stability of the coatings permitted the generation of mass spectra without interfering peaks originating from the coating polymers. The high EOF generated by the coatings facilitated the maintenance of a stable spray when coupling to ESI-MS, and a stable CZE current when working with a sheath flow-assisted analyte deposition onto MALDI targets, respectively. In conclusion, CZE-MS could be demonstrated as a robust complementary method to capillary RP-HPLC-MS in combination with both soft-ionization techniques, ESI and MALDI, generally, and particularly in the context of glycopeptide analysis.     

Protein structure and dynamics studied by mass spectrometry: H/D exchange, hydroxyl radical labeling, and related approaches

Mass spectrometry (MS) plays a central role in studies on protein structure and dynamics. This review highlights some of the recent developments in this area, with focus on applications involving the use of electrospray ionization (ESI) MS. Although this technique involves the transformation of analytes into highly nonphysiological species (desolvated gas-phase ions in the vacuum), ESI-MS can provide detailed insights into the solution-phase behavior of proteins. Notably, the ionization process itself occurs in a structurally sensitive manner. An increased degree of solution-phase unfolding is correlated with a higher level of protonation. Also, ESI allows the transfer of intact noncovalent complexes into the gas phase, thereby yielding information on binding partners, stoichiometries, and even affinities. A particular focus of this article is the use of hydrogen/deuterium exchange (HDX) methods and hydroxyl radical (.OH) labeling for monitoring dynamic and structural aspect of solution-phase proteins. Conceptual similarities and differences between the two methods are discussed. We describe a simple method for the computational simulation of protein HDX patterns, a tool that can be helpful for the interpretation of isotope exchange data recorded under mixed EX1/EX2 conditions. Important aspects of .OH labeling include a striking dependence on protein concentration, and the tendency of commonly used solvent additives to act as highly effective radical scavengers. If not properly controlled, both of these factors may lead to experimental artifacts.     

Triple quadrupole mass spectrometry as applied to flame diagnostics: study of the C2H4/N2O/Ar flame

A recently developed research apparatus for characterization of low-pressure premixed flames has been developed and was used to characterize the C₂H₄/N₂O/Ar flame at 20 torr. This instrument incorporates several diagnostic techniques in one apparatus so that individual techniques can be quantitatively compared and the usable detection range (both in terms of resolution and species detection) expanded. Results discussed in this report include mass analysis by triple quadrupole mass spectrometer and temperature measurement by thermocouple. Concentration profiles in the one-dimensional flame include CO, N₂, and C₂H₄, at nominal m/z 28 as well as CO₂ and N₂O at m/z 44.     

Measurements by gas chromatography/pyrolysis/mass spectrometry: fundamental conditions in (2)H/(1)H isotope ratio analysis

Gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS) is a relatively new method for on-line determination of (2)H/(1)H isotope ratios. The influence of different parameters on the (2)H/(1)H isotope ratios obtained in GC/P/IRMS has been thoroughly studied using several flavor compounds, such as 5-nonanone, linalool, menthol, linalyl acetate and 4-decanolide. The requirement of "conditioning" the pyrolysis reactor to obtain reliable delta(2)H(V-SMOW) values is discussed. Furthermore, the influence of the carrier gas flow of the gas chromatograph on the completeness of pyrolysis and subsequently on the delta(2)H(V-SMOW) values is investigated in detail. The linear range of the compounds investigated is determined. The results show that calibrating the GC/P/IRMS system with secondary standard substances is absolutely necessary in order to obtain reliable delta(2)H(V-SMOW) values. In view of interlaboratory comparability, validation procedures are recommended.     

H/D exchange under mild conditions in arenes and unactivated alkanes with C6D6 and D2O using rigid,electron-rich iridium PCP pincer complexes

The synthesis and characterization of an iridium polyhydride complex (Ir-H4) supported by an electron-rich PCP framework is described. This complex readily loses molecular hydrogen allowing for rapid room temperature hydrogen isotope exchange (HIE) at the hydridic positions and the α-C–H site of the ligand with deuterated solvents such as benzene-d₆, toluene-d₈ and THF-d₈. The removal of 1–2 equivalents of molecular H₂ forms unsaturated iridium carbene trihydride (Ir-H3) or monohydride (Ir-H) compounds that are able to create further unsaturation by reversibly transferring a hydride to the ligand carbene carbon. These species are highly active hydrogen isotope exchange (HIE) catalysts using C₆D₆ or D₂O as deuterium sources for the deuteration of a variety of substrates. By modifying conditions to influence the Ir-Hn speciation, deuteration levels can range from near exhaustive to selective only for sterically accessible sites. Preparative level deuterations of select substrates were performed allowing for procurement of >95% deuterated compounds in excellent isolated yields; the catalyst can be regenerated by treatment of residues with H₂ and is still active for further reactions.

The synthesis and characterization of an iridium polyhydride complex (Ir-H4) supported by an electron-rich PCP framework and capable of mild hydrogen/deuterium exchange catalysis is described.     

Capillary electrophoresis coupled to inductively coupled plasma mass spectrometry (CE/ICP-MS) and to electrospray ionization mass spectrometry (CE/ESI-MS): An approach for maximum species information in speciation of selenium

On-line hyphenations consisting of a separation and a detection step are one of the most efficient techniques for identification and characterization of metal containing species. The high resolution power of capillary electrophoresis (CE) is used for the separation of three selenium species, whereas either electrospray ionization mass spectrometry (ESI-MS) or inductively coupled plasma mass spectrometry (ICP-MS) are taken for molecule or element specific detection. This work gives an overview about the possibilities and limitations, when using the two hyphenated systems for speciation investigations. In order to show the power of the two complementary techniques, a CZE method using 5% acetic acid as background electrolyte was applied to the separation of selenomethionine (SeM), selenocystine (SeC) and selenocystamine (SeCM). Depending on the species and the element, the detection limits of the CE/ICP-MS hyphenated system are up to 10² to 10³ times better than that for the CE/ESI-MS system.     

Conformational studies of the robust 2-Cys peroxiredoxin Salmonella typhimurium AhpC by solution phase hydrogen/deuterium (H/D) exchange monitored by electrospray ionization mass spectrometry

This is the first comprehensive HX-MS study of a "robust" 2-Cys peroxiredoxin (Prx), namely Salmonella typhimurium AhpC (StAhpC). Prx proteins control intracellular peroxide levels and are abundant antioxidant proteins in eukaryotes, archaea and bacteria. Crystal structural analyses and structure/activity studies of several bacterial and mammalian 2-Cys Prxs have revealed that the activity of 2-Cys Prxs is regulated by redox-dependent oligmerization and a sensitivity of the active site cysteine residue to overoxidation. The propensity to overoxidation is linked to the conformational flexibility of the peroxidatic active site loop. The HX-MS results emphasize the modulation of the conformational motility of the active site loop by disulfide formation. To obtain information on the conformational impact of decamer formation on the active site loop motility, mutants with Thr77 substituted by Ile, a decamer-disrupting mutation or by Val, a decamer-stabilizing mutation, were studied. For the isoleucine mutant, enhanced mobility was observed for regions encompassing the α4 helix located in the dimer-dimer interface and regions surrounding the peroxidatic loop. In contrast, the T77V mutation resulted in an increase in conformational stability in most regions of the protein except for the active site loop and the region encompassing the resolving cysteine.     

Using gas chromatography/isotope ratio mass spectrometry to determine the fractionation factor for H2 production by hydrogenases

Hydrogenases catalyze the reversible formation of H(2), and they are key enzymes in the biological cycling of H(2). H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H(2) production processes, but there are several obstacles that have thus far limited the application of this tool. Here, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme-catalyzed H(2) evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. In addition, a custom-designed high-throughput gas chromatograph/isotope ratio mass spectrometer is employed to measure the isotope ratio of the H(2). Using our new approach, we determined that the fractionation factor for H(2) production by the [NiFe]-hydrogenase from Desulfovibrio fructosovorans is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H(2) evolution. Potential applications of this newly developed method are discussed.     

Review: Current applications and challenges for liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS)

High-precision isotope analysis is recognized as an essential research tool in many fields of study. Until recently, continuous flow isotope ratio mass spectrometry (CF-IRMS) was available via an elemental analyzer or a gas chromatography inlet system for compound-specific analysis of light stable isotopes. In 2004, however, an interface that coupled liquid chromatography with IRMS (LC/IRMS) became commercially available for the first time. This brought the capability for new areas of application, in particular enabling compound-specific δ(13)C analysis of non-volatile, aqueous soluble, compounds from complex mixtures. The interface design brought with it several analytical constraints, however, in particular a lack of compatibility with certain types of chromatography as well as limited flow rates and mobile phase compositions. Routine LC/IRMS methods have, however, been established for measuring the δ(13)C isotopic ratios of underivatized individual compounds for application in archeology, nutrition and physiology, geochemistry, hydrology, soil science and food authenticity. Seven years after its introduction, we review the technical advances and constraints, methodological developments and new applications of liquid chromatography coupled to isotope ratio mass spectrometry.