Aspergillus niger Prolyl Endoprotease for Hydrogen-Deuterium Exchange Mass Spectrometry and Protein Structural Studies.
ABSTRACT: 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: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 0Â°C and pH 2.5. Very few proteases are known to withstand such harsh conditions, with pepsin being the best-known exception, even though its activity is also strongly reduced at 0Â°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 its ability to cleave 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 (PRPs) or proteins containing Pro-rich binding domains involved in cellular signaling.
Project description:The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from >30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5-6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.
Project description:Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5-10 years. The predominant technology requires fast digestion at pH 2-3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to "forbidden" residues K, R, H, and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to "tune" the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the nonhomologous end-joining DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their binding partners.
Project description:The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (?-Lg) and alpha-lactalbumin (?-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/?g test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach.
Project description:The functional properties and adipogenesis inhibitory activity of quinoa protein hydrolysates, prepared using papain, pepsin, and pancreatin for 0, 30, 60, 90, and 120 min, were studied. For these three kinds of proteases, the solubility of the hydrolysates significantly increased with the increasing DH in pH range of 3-8, while the EAI and ESI of these hydrolysates significantly decreased during hydrolysis. The anti-inflammatory activity of these protein hydrolysates was measured. All of these protein hydrolysates showed high anti-inflammatory activity. However, there was no significant difference in anti-inflammatory activity between protein hydrolysates and total protein from quinoa. These protein hydrolysates also inhibited lipid accumulation during differentiation within the range of concentrations of 0-1,600 ?g/ml, which exerted no cytotoxicity toward 3T3-L1 cells. The protein hydrolysates from quinoa prepared using pepsin for 120 min (PEP-120) had the highest activity with an IC50 value of 786.58 ?g/ml. Moreover, LC-MS/MS analysis of PEP-120 showed that five main bioactive peptides, which have been demonstrated to have ACE inhibitor, antioxidant, and antithrombotic activities, were present in PEP-120. In addition, gene expression and Western blot analysis revealed that PEP-120 suppressed the 3T3-L1 cell differentiation through the peroxisome proliferator-activated receptor ? (PPAR?) pathway.
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:Membrane proteins are currently the most common targets for pharmaceuticals. However, characterization of their structural dynamics by hydrogen/deuterium exchange mass spectrometry (HDX-MS) is sparse due to insufficient automated methods to handle full-length membrane proteins in lipid bilayers. Additionally, membrane lipids used to mimic the membrane environment and to solubilize membrane proteins can impair chromatography performance and cause ion suppression in the mass spectrometer. The workflow discussed herein advances HDX-MS capabilities and other MS applications for membrane proteins by providing a fully automated method for HDX-MS analysis based on a phospholipid removal scheme compatible with robotic handling. Phospholipids were depleted from protein samples by the addition of zirconium oxide beads, which were subsequently removed by inline filtration using syringeless nanofilters. To demonstrate this method, single-pass transmembrane protein Fc?RIIa (CD32a) expressed into liposomes was used. Successful depletion of phospholipids ensured optimal liquid-chromatography-mass-spectrometry performance, and measurement of peptides from the transmembrane domain of Fc?RIIa indicated phospholipids associated with this region were either not present or did not shield the transmembrane domain from digestion by pepsin. Furthermore, amino acid sequence coverage provided by this method was suitable to enable future measurement of structural dynamics of ectodomain, transmembrane domain, and endodomain of Fc?RIIa. Moreover, this method is the first to enable fully automated HDX-MS on full-length transmembrane proteins in lipid bilayers, a notable advancement to facilitate understanding of membrane proteins, development of pharmaceuticals, and characterization for regulatory agencies.
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:eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5? UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.
Project description:Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concentration) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clinical relevance. Through two rounds of iterative mutagenesis and analysis, variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, especially in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and analytical cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacologically significant improvements in gluten detoxification activity.