Online, high-pressure digestion system for protein characterization by hydrogen/deuterium exchange and mass spectrometry.
ABSTRACT: The rapid and complete digestion of proteins is important when protein characterization by hydrogen-deuterium exchange (HDX) is coupled with mass spectrometry. We developed a single-pump, online, high-pressure digestion system that relies on UPLC technology to aid in the digestion of proteins. Two model proteins, amyloid beta-peptide 1-42 (Abeta 1-42) and an HIV-1 capsid mutant protein (NBSA), were used to demonstrate the efficacy of the high-pressure system. Both model proteins readily aggregate and are difficult to digest under normal conditions. Our high-pressure system successfully digests these proteins into small, overlapping peptides. The extra information afforded by overlapping peptides allows us to pinpoint HDX protection to protein segments smaller than the digested peptide. The calculated average segment length (ASL) for both model proteins decreased by 2-fold for high-pressure digestion compared to digestion at ambient pressure.
Project description:Characterization of interactions between proteins and other molecules is crucial for understanding the mechanisms of action of biological systems and, thus, drug discovery. An increasingly useful approach to mapping these interactions is measurement of hydrogen/deuterium exchange (HDX) using mass spectrometry (HDX-MS), which measures the time-resolved deuterium incorporation of peptides obtained by enzymatic digestion of the protein. Comparison of exchange rates between apo- and ligand-bound conditions results in a mapping of the differential HDX (?HDX) of the ligand. Residue-level analysis of these data, however, must account for experimental error, sparseness, and ambiguity due to overlapping peptides. Here, we propose a Bayesian method consisting of a forward model, noise model, prior probabilities, and a Monte Carlo sampling scheme. This method exploits a residue-resolved exponential rate model of HDX-MS data obtained from all peptides simultaneously, and explicitly models experimental error. The result is the best possible estimate of ?HDX magnitude and significance for each residue given the data. We demonstrate the method by revealing richer structural interpretation of ?HDX data on two nuclear receptors: vitamin D-receptor (VDR) and retinoic acid receptor gamma (ROR?). The method is implemented in HDX Workbench and as a standalone module of the open source Integrative Modeling Platform.
Project description:Proteolyzed peptides provide the basis for mass-analyzed hydrogen/deuterium exchange (HDX) for mapping solvent access to various segments of solution-phase proteins. Aspergillus saitoi protease type XIII and porcine pepsin can generate peptides of overlapping sequences and high sequence coverage. However, if disulfide bonds are present, proteolysis can be severely limited, particularly in the vicinity of the disulfide linkage(s). Disulfide bonds cannot be reduced before or during the H/D exchange reaction without affecting the protein higher-order structure. Here, we demonstrate simultaneous quench/digestion/reduction following H/D exchange, for subsequent mass analysis. Proteolysis is conducted in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) and urea, and all other steps of the H/D exchange and analysis are maintained. This method yields dramatically increased sequence coverage and localization of solvent-exposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.
Project description:Hydrogen-deuterium exchange mass spectrometry is an important method for protein structure-function analysis. The bottom-up approach uses protein digestion to localize deuteration to higher resolution, and the essential measurement involves centroid mass determinations on a very large set of peptides. In the course of evaluating systems for various projects, we established two (HDX-MS) platforms that consisted of a FT-MS and a high-resolution QTOF mass spectrometer, each with matched front-end fluidic systems. Digests of proteins spanning a 20-110 kDa range were deuterated to equilibrium, and figures-of-merit for a typical bottom-up (HDX-MS) experiment were compared for each platform. The Orbitrap Velos identified 64% more peptides than the 5600 QTOF, with a 42% overlap between the two systems, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the QTOF, depending on the Orbitrap resolution setting. However, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate, because of destructive interference arising from mismatches in elemental mass defects. This is shown through the analysis of the peptides common to both platforms, where deuteration values can be as low as 35% of the expected values, depending on FT-MS resolution, peptide length and charge state. These findings are supported by simulations of Orbitrap transients, and highlight that caution should be exercised in deriving centroid mass values from FT transients that do not support baseline separation of the full isotopic composition.
Project description:To monitor the structural integrity of therapeutic proteins, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is increasingly utilized in the pharmaceutical industry. The successful outcome of HDX-MS analyses depends on the sample preparation conditions, which involve the rapid digestion of proteins at 0 °C and pH 2.5. Very few proteases are able to withstand such harsh conditions, with pepsin being the best-known exception, even though its activity is also strongly reduced at 0 °C. Here, we evaluate the usage of a prolyl endopeptidase from Aspergillus niger (An-PEP) for HDX-MS. What makes this protease very attractive is that it cleaves preferentially the hardest to digest amino acid, proline. To our surprise, and in contrast to previous reports, An-PEP activity was found optimal around pH 2.5 and could be further enhanced by urea up to 40%. Under typical HDX-MS conditions and using small amounts of enzyme, An-PEP generated an equivalent number of peptides as pepsin, as exemplified by using the two model systems tetrameric human hemoglobin (Hb) and human IgG4. Interestingly, because An-PEP peptides are shorter than pepsin-generated peptides, higher sequence resolution could be achieved, especially for Pro-containing protein regions in the alpha subunit of Hb, revealing new protected Hb regions that were not observed with pepsin. Due to its Pro-preference and resistance to low pH, we conclude that An-PEP is an archetype enzyme for HDX-MS, highly complementary to pepsin, and especially promising for structural studies on Pro-rich proteins or proteins containing Pro-rich binding domains involved in cellular signaling.
Project description:The local effects of hydration on myoglobin (Mb) in solid matrices containing mannitol or sucrose (1:1 w/w, protein:additive) were mapped using hydrogen-deuterium exchange with mass spectrometric analysis (HDX-MS) at 5 °C and compared to solution controls. Solid powders were exposed to D?O(g) at controlled activity (a(w)) followed by reconstitution and analysis of the intact protein and peptides produced by pepsin digestion. HDX varied with matrix type, a(w), and position along the protein backbone. HDX was less in sucrose matrices than in mannitol matrices at all a(w) while the difference in solution was negligible. Differences in HDX in the two matrices were detectable despite similarities in their bulk water content. The extent of exchange in solids is proposed as a measure of the hydration of exchangeable amide groups, as well as protein conformation and dynamics; pepsin digestion allows these effects to be mapped with peptide-level resolution.
Project description:Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry offers a rapid method to study protein conformations and protein-protein interactions. Pepsin is usually used to digest proteins in HDX and is known for lack of cleavage specificity. To improve digestion efficiency and specificity, we have optimized digestion conditions and cleavage preferences for pepsin and protease type XIII from Aspergillus saitoi. A dilution series of the proteases was used to determine the digestion efficiency for several test proteins. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII from Rhizhopus. Furthermore, protease type XIII exhibited much less self-digestion than pepsin and thus is superior for HDX experiments. Many highly overlapped segments from protease type XIII and pepsin digestion, combined with high-resolution FTICR mass spectrometry, provide high sequence resolution (to as few as one or two amino acids) for the assignment of amide hydrogen exchange rate. Our H/D exchange results correlate well with the secondary and tertiary structure of myoglobin. Such assignments of highly overlapped fragments promise to greatly enhance the accuracy and sequence resolution for determining conformational differences resulting from ligand binding or protein-protein interactions.
Project description:Pepsin was immobilized on ethyl-bridged hybrid (BEH) particles, and digestion performance was evaluated in a completely online format, with the specific intent of using the particles for hydrogen-deuterium exchange mass spectrometry (HDX MS) experiments. Because the BEH particles are mechanically strong, they could withstand prolonged, continuous high-pressure at 10,000 psi. Online digestion was performed under isobaric conditions with continuous solvent flow, in contrast to other approaches where the pressure or flow is cycled. As expected, digestion efficiency at 10,000 psi was increased and reproducibly produced more peptic peptides versus digestion at 1000 psi. Prototype columns made with the BEH pepsin particles exhibited robust performance, and deuterium back-exchange was similar to that of other immobilized pepsin particles. These particles can be easily incorporated in existing HDX MS workflows to provide more peptide coverage in experiments where fast, efficient, and reproducible online pepsin digestion is desired.
Project description:Hydrogen deuterium exchange mass spectrometry (HDX MS) reports on the conformational landscape of proteins by monitoring the exchange between backbone amide hydrogen atoms and deuterium in the solvent. To maintain the label for analysis, quench conditions of low temperature and pH are required during the chromatography step performed after protease digestion but before mass spectrometry. Separation at 0°C is often chosen as this is the temperature where the most deuterium can be recovered without freezing of the typical water and acetonitrile mobile phases. Several recent reports of separations at subzero Celsius emphasize the promise for retaining more deuterium and using a much longer chromatographic gradient or direct infusion time. Here we present the construction and validation of a modified Waters nanoACQUITY HDX manager with a third temperature-controlled zone for peptide separations at subzero temperatures. A new Peltier-cooled door replaces the door of a traditional main cooling chamber and the separations and trapping column are routed through the door housing. To prevent freezing, 35% methanol is introduced post online digestion. No new pumps are required and online digestion is performed as in the past. Subzero separations, using conventional HPLC column geometry of 3? m particles in a 1×50mm column, did not result in major changes to chromatographic efficiency when lowering the temperature from 0 to -20°C. There were significant increases in deuterium recovery for both model peptides and biologically relevant protein systems. Given the higher levels of deuterium recovery, expanded gradient programs can be used to allow for higher chromatographic peak capacity and therefore the analysis of larger and more complex proteins and systems.
Project description:We describe a platform utilizing two methods based on hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 ?M for the three different regions, giving an overall binding affinity of 7.4 ?M. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, ?C1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein-ligand interactions.
Project description:Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, nonbinding control peptides, and statistical analysis of replicate data. We show that HDX-MS can identify regions in the two epigenetic regulator proteins KDM4C and KDM1A that are perturbed through weak interactions with PD-identified peptides. Two peptides cause reduced HDX on opposite sides of the active site of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches.