Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.

Conformationally Restricted Glycopeptide Backbone Inhibits Gas-Phase H/D Scrambling between Glycan and Peptide Moieties.

Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS). However, it is, in most cases, not possible to extract information about glycan conformation and dynamics due to the general difficulty of separating the deuterium content of the glycan from that of the peptide (in particular, for O-linked glycans). Here, we investigate whether the fragmentation of protonated glycopeptides by collision-induced dissociation (CID) can be used to determine the solution-specific deuterium content of the glycan. Central to this concept is that glycopeptides can undergo a facile loss of glycans upon CID, thereby allowing for the determination of their masses. However, an essential prerequisite is that hydrogen and deuterium (H/D) scrambling can be kept in check. Therefore, we have measured the degree of scrambling upon glycosidic bond cleavage in glycopeptides that differ in the conformational flexibility of their backbone and glycosylation pattern. Our results show that complete scrambling precedes the glycosidic bond cleavage in normal glycopeptides derived from a glycoprotein; i.e., all labile hydrogens have undergone positional randomization prior to loss of the glycan. In contrast, the glycosidic bond cleavage occurs without any scrambling in the glycopeptide antibiotic vancomycin, reflecting that the glycan cannot interact with the peptide moiety due to a conformationally restricted backbone as revealed by molecular dynamics simulations. Scrambling is also inhibited, albeit to a lesser degree, in the conformationally restricted glycopeptides ristocetin and its pseudoaglycone, demonstrating that scrambling depends on an intricate interplay between the flexibility and proximity of the glycan and the peptide backbone.

Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS.

Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

Epitope mapping of a blood-brain barrier crossing antibody targeting the cysteine-rich region of IGF1R using hydrogen-exchange mass spectrometry enabled by electrochemical reduction.

Pathologies of the central nervous system impact a significant portion of our population, and the delivery of therapeutics for effective treatment is challenging. The insulin-like growth factor-1 receptor (IGF1R) has emerged as a target for receptor-mediated transcytosis, a process by which antibodies are shuttled across the blood-brain barrier (BBB). Here, we describe the biophysical characterization of VHH-IR4, a BBB-crossing single-domain antibody (sdAb). Binding was confirmed by isothermal titration calorimetry and an epitope was highlighted by surface plasmon resonance that does not overlap with the IGF-1 binding site or other known BBB-crossing sdAbs. The epitope was mapped with a combination of linear peptide scanning and hydrogen-deuterium exchange mass spectrometry (HDX-MS). IGF1R is large and heavily disulphide bonded, and comprehensive HDX analysis was achieved only through the use of online electrochemical reduction coupled with a multiprotease approach, which identified an epitope for VHH-IR4 within the cysteine-rich region (CRR) of IGF1R spanning residues W244-G265. This is the first report of an sdAb binding the CRR. We show that VHH-IR4 inhibits ligand induced auto-phosphorylation of IGF1R and that this effect is mediated by downstream conformational effects. Our results will guide the selection of antibodies with improved trafficking and optimized IGF1R binding characteristics.

PNGase H + variant from Rudaea cellulosilytica with improved deglycosylation efficiency for rapid analysis of eukaryotic N-glycans and hydrogen deuterium exchange mass spectrometry analysis of glycoproteins.

The analysis of glycoproteins and the comparison of protein N-glycosylation from different eukaryotic origins require unbiased and robust analytical workflows. The structural and functional analysis of vertebrate protein N-glycosylation currently depends extensively on bacterial peptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidases (PNGases), which are indispensable enzymatic tools in releasing asparagine-linked oligosaccharides (N-glycans) from glycoproteins. So far, only limited PNGase candidates are available for N-glycans analysis, and particularly the analysis of plant and invertebrate N-glycans is hampered by the lack of suitable PNGases. Furthermore, liquid chromatography-mass spectrometry (LC-MS) workflows, such as hydrogen deuterium exchange mass spectrometry (HDX-MS), require a highly efficient enzymatic release of N-glycans at low pH values to facilitate the comprehensive structural analysis of glycoproteins. Herein, we describe a previously unstudied superacidic bacterial N-glycanase (PNGase H+ ) originating from the soil bacterium Rudaea cellulosilytica (Rc), which has significantly improved enzymatic properties compared to previously described PNGase H+ variants. Active and soluble recombinant PNGase Rc was expressed at a higher protein level (3.8-fold) and with higher specific activity (~56% increase) compared to the currently used PNGase H+ variant from Dyella japonicum (Dj). Recombinant PNGase Rc was able to deglycosylate the glycoproteins horseradish peroxidase and bovine lactoferrin significantly faster than PNGase Dj (10 min vs. 6 h). The versatility of PNGase Rc was demonstrated by releasing N-glycans from a diverse array of samples such as peach fruit, king trumpet mushroom, mouse serum, and the soil nematode Caenorhabditis elegans. The presence of only two disulfide bonds shown in the AlphaFold protein model (so far all other superacidic PNGases possess more disulfide bonds) could be corroborated by intact mass- and peptide mapping analysis and provides a possible explanation for the improved recombinant expression yield of PNGase Rc.

The dopamine transporter antiports potassium to increase the uptake of dopamine.

The dopamine transporter facilitates dopamine reuptake from the extracellular space to terminate neurotransmission. The transporter belongs to the neurotransmitter:sodium symporter family, which includes transporters for serotonin, norepinephrine, and GABA that utilize the Na+ gradient to drive the uptake of substrate. Decades ago, it was shown that the serotonin transporter also antiports K+, but investigations of K+-coupled transport in other neurotransmitter:sodium symporters have been inconclusive. Here, we show that ligand binding to the Drosophila- and human dopamine transporters are inhibited by K+, and the conformational dynamics of the Drosophila dopamine transporter in K+ are divergent from the apo- and Na+-states. Furthermore, we find that K+ increases dopamine uptake by the Drosophila dopamine transporter in liposomes, and visualize Na+ and K+ fluxes in single proteoliposomes using fluorescent ion indicators. Our results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K+ on other transporters in this pharmacologically important family.

Probing the Impact of Temperature and Substrates on the Conformational Dynamics of the Neurotransmitter:Sodium symporter LeuT.

The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure-function relationships of NSS family proteins. With hydrogen-deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperatures (25 °C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 - 95 °C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45 °C and 60 °C). At these elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25 °C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. We have further investigated the conformational impact of binding the efficiently transported substrate alanine (Ala) relative to the much slower transported substrate leucine (Leu). Comparing the HDX of the Ala-bound versus Leu-bound state of LeuT, we observe distinct differences that could explain the faster transport rate (kcat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of Ala-bound LeuT . Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.

Conformational dynamics of free and membrane-bound human Hsp70 in model cytosolic and endo-lysosomal environments.

The binding of the major stress-inducible human 70-kDa heat shock protein (Hsp70) to the anionic phospholipid bis-(monoacylglycero)-phosphate (BMP) in the lysosomal membrane is crucial for its impact on cellular pathology in lysosomal storage disorders. However, the conformational features of this protein-lipid complex remain unclear. Here, we apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe the dynamics of the full-length Hsp70 in the cytosol and its conformational changes upon translocation into lysosomes. Using wild-type and W90F mutant proteins, we also map and discriminate the interaction of Hsp70 with BMP and other lipid components of the lysosomal membrane. We identify the N-terminal of the nucleotide binding domain (residues 87-118) as the primary orchestrator of BMP interaction. We show that the conformation of this domain is significantly reorganized in the W90F mutant, explaining its inability to stabilize lysosomal membranes. Overall, our results reveal important new molecular details of the protective effect of Hsp70 in lysosomal storage diseases, which, in turn, could guide future drug development.

Hydrogen/Deuterium Exchange Mass Spectrometry with Integrated Electrochemical Reduction and Microchip-Enabled Deglycosylation for Epitope Mapping of Heavily Glycosylated and Disulfide-Bonded Proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a recognized method to study protein conformational dynamics and interactions. Proteins encompassing post-translational modifications (PTMs), such as disulfide bonds and glycosylations, present challenges to HDX-MS, as disulfide bond reduction and deglycosylation is often required to extract HDX information from regions containing these PTMs. In-solution deglycosylation with peptide-N4-(N-acetyl-ß-d-glucosaminyl)-asparagine amidase A (PNGase A) or PNGase H+ combined with chemical reduction using tris-(2-carboxyethyl)phosphine (TCEP) has previously been used for HDX-MS analysis of disulfide-linked glycoproteins. However, this workflow requires extensive manual sample preparation and consumes large amounts of enzyme. Furthermore, large amounts of TCEP and glycosidases often result in suboptimal liquid chromatography-mass spectrometry (LC-MS) performance. Here, we compare the in-solution activity of PNGase A, PNGase H+, and the newly discovered PNGase Dj under quench conditions and immobilize them onto thiol-ene microfluidic chips to create HDX-MS-compatible immobilized microfluidic enzyme reactors (IMERs). The IMERS retain deglycosylation activity, also following repeated use and long-term storage. Furthermore, we combine a PNGase Dj IMER, a pepsin IMER, and an electrochemical cell to develop an HDX-MS setup capable of efficient online disulfide-bond reduction, deglycosylation, and proteolysis. We demonstrate the applicability of this setup by mapping the epitope of a monoclonal antibody (mAb) on the heavily disulfide-bonded and glycosylated sema-domain of the tyrosine-protein kinase Met (SD c-Met). We achieve near-complete sequence coverage and extract HDX data to identify regions of SD c-Met involved in mAb binding. The described methodology thus presents an integrated and online workflow for improved HDX-MS analysis of challenging PTM-rich proteins.

Hydrogen-Deuterium Exchange Mass Spectrometry with Integrated Size-Exclusion Chromatography for Analysis of Complex Protein Samples.

The growing use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) for studying membrane proteins, large protein assemblies, and highly disulfide-bonded species is often challenged by the presence in the sample of large amounts of lipids, protein ligands, and/or highly ionizable reducing agents. Here, we describe how a short size-exclusion chromatography (SEC) column can be integrated with a conventional temperature-controlled HDX-MS setup to achieve fast and online removal of unwanted species from the HDX sample prior to chromatographic separation and MS analysis. Dual-mode valves permit labeled proteins eluting after SEC to be directed to the proteolytic and chromatographic columns, while unwanted sample components are led to waste. The SEC-coupled HDX-MS method allows analyses to be completed with lower or similar back-exchange compared to conventional experiments. We demonstrate the suitability of the method for the analysis of challenging protein samples, achieving efficient online removal of lipid components from protein-lipid systems, depletion of an antibody from an antigen during epitope mapping, and elimination of MS interfering compounds such as tris(2-carboxyethyl)phosphine (TCEP) during HDX-MS analysis of a disulfide-bonded protein. The implementation of the short SEC column to the conventional HDX-MS setup is straightforward and could be of significant general utility during the HDX-MS analysis of complex protein states.

Epitope Mapping of Polyclonal Antibodies by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Epitope mapping of antibodies (Abs) is crucial for understanding adaptive immunity, as well as studying the mode of action of therapeutic antibodies and vaccines. Especially insights into the binding of the entire polyclonal antibody population (pAb) raised upon vaccination would be of unique value to vaccine development. However, very few methods for epitope mapping can tolerate the complexity of a pAb sample. Here we show how hydrogen-deuterium exchange mass spectrometry (HDX-MS) can be used to map epitopes recognized by pAb samples. Our approach involves measuring the HDX of the antigen in absence or presence of varied amounts of pAbs, as well as dissociating additives. We apply the HDX-MS workflow to pAbs isolated from rabbit immunized with factor H-binding protein (fHbp), a Neisseria meningitidis vaccine antigen. We identify four immunogenic regions located on the N- and C-terminal region of fHbp and provide insights into the relative abundance and avidity of epitope binding Abs present in the sample. Overall, our results show that HDX-MS can provide a unique and relatively fast method for revealing the binding impact of the entire set of pAbs present in blood samples after vaccination. Such information provides a rare view into effective immunity and can guide the design of improved vaccines against viruses or bacteria.

Revealing the Dynamic Allosteric Changes Required for Formation of the Cysteine Synthase Complex by Hydrogen-Deuterium Exchange MS.

CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function. Being absent in mammals, the two enzymes and their complex are attractive targets for antibacterial drugs. We have used hydrogen/deuterium exchange MS to unveil how complex formation affects the conformational dynamics of CysK and CysE. Our results support a model where CysE is present in solution as a dimer of trimers, and each trimer can bind one CysK homodimer. When CysK binds to one CysE monomer, intratrimer allosteric communication ensures conformational and dynamic symmetry within the trimer. Furthermore, a long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation. These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design.

Probing the Conformational Dynamics of Affinity-Enhanced T Cell Receptor Variants upon Binding the Peptide-Bound Major Histocompatibility Complex by Hydrogen/Deuterium Exchange Mass Spectrometry.

Binding of the T cell receptor (TCR) to its cognate, peptide antigen-loaded major histocompatibility complex (pMHC) is a key interaction for triggering T cell activation and ultimately elimination of the target cell. Despite the importance of this interaction for cellular immunity, a comprehensive molecular understanding of TCR specificity and affinity is lacking. We conducted hydrogen/deuterium exchange mass spectrometry (HDX-MS) analyses of individual affinity-enhanced TCR variants and clinically relevant pMHC class I molecules (HLA-A*0201/NY-ESO-1157-165) to investigate the causality between increased binding affinity and conformational dynamics in TCR-pMHC complexes. Differential HDX-MS analyses of TCR variants revealed that mutations for affinity enhancement in TCR CDRs altered the conformational response of TCR to pMHC ligation. Improved pMHC binding affinity was in general observed to correlate with greater differences in HDX upon pMHC binding in modified TCR CDR loops, thereby providing new insights into the TCR-pMHC interaction. Furthermore, a specific point mutation in the ß-CDR3 loop of the NY-ESO-1 TCR associated with a substantial increase in binding affinity resulted in a substantial change in pMHC binding kinetics (i.e., very slow kon, revealed by the detection of EX1 HDX kinetics), thus providing experimental evidence for a slow induced-fit binding mode. We also examined the conformational impact of pMHC binding on an unrelated TRAV12-2 gene-encoded TCR directed against the immunodominant MART-126-35 cancer antigen restricted by HLA-A*0201. Our findings provide a molecular basis for the observed TRAV12-2 gene bias in natural CD8+ T cell-based immune responses against the MART-1 antigen, with potential implications for general ligand discrimination and TCR cross-reactivity processes.

Epitope and Paratope Mapping by HDX-MS Combined with SPR Elucidates the Difference in Bactericidal Activity of Two Anti-NadA Monoclonal Antibodies.

Characterization of antigen-antibody interactions is crucial for understanding antibody-mediated protection against pathogens, biopharmaceutical development, as well as evaluation of the immune response post vaccination. Bexsero is a multicomponent vaccine against Neisseria meningitidis serogroup B in which one of the key vaccine antigens is Neisserial adhesin A (NadA), a trimeric coiled-coil protein. Two NadA-specific monoclonal antibodies (mAbs) isolated from Bexsero-vaccinated individuals have been shown to have similar binding affinity and appear to recognize a similar antigen region, yet only one of the mAbs is bactericidal. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to perform an in-depth study of the interaction of the two mAbs with NadA antigen using a combined epitope and paratope mapping strategy. In addition, we use surface plasmon resonance (SPR) to investigate the stoichiometry of the binding of the two mAbs to NadA. While epitope mapping only identifies a clear binding impact of one of the mAbs on NadA, the paratope mapping analyses shows that both mAbs are binding to NadA through several complementarity determining regions spanning both heavy and light chains. Our results highlight the advantage of combined epitope and paratope mapping HDX-MS experiments and supporting biochemical experiments to characterize antigen-antibody interactions. Through this combined approach, we provide a rationale for how the binding stoichiometry of the two mAbs to the trimeric NadA antigen can explain the difference in bactericidal activity of the two mAbs.

Thiol-ene microfluidic chip for fast on-chip sample clean-up, separation and ESI mass spectrometry of peptides and proteins.

Mass spectrometry (MS) is a key technology for sensitive and high-resolution mass analysis of peptides and proteins. Sample clean-up and chromatographic separation is typically performed prior to MS analysis to limit adduct formation and ionization suppression. Usually, this requires a high-pressure LC pump system equipped with expensive metal chromatographic columns placed in-line of an electrospray ionization (ESI) source. Microfluidic devices coupled to MS have gained considerable attention, due to the promise of low manufacturing costs, low sample consumption and channels with a high surface area to volume ratio and tailorable functional groups. Here, we describe a thiol-ene microfluidic chip capable of fast chromatographic sample clean-up, concentration, and separation of complex protein and peptide mixtures with direct on-chip ESI. On-chip reversed-phase chromatography (RPC) was performed through an in-situ polymerized monolith frit for retaining inexpensive commercially available reversed-phase (RP) spherical particles, while on-chip ESI is achieved through an emitter monolithically implemented by precision micro milling. The on-chip integration of both RPC and ESI emitter allowed for a minimization of dead-volumes and enables very fast sample clean-up, efficient ionization, and mass analysis of peptides and proteins from complex matrices.

Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H+, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H+ retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H+ allowed the extraction of HDX data from all five glycosylated regions of the serpin α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H+ provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H+ for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.

Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis.

BACKGROUND: Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. METHODS: Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. RESULTS: The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

Discovery of Highly Active Recombinant PNGase H+ Variants Through the Rational Exploration of Unstudied Acidobacterial Genomes.

Peptide-N 4-(N-acetyl-ß-glucosaminyl) asparagine amidases (PNGases, N-glycanases, EC 3.5.1.52) are indispensable tools in releasing N-glycans from glycoproteins. So far, only a limited number of PNGase candidates are available for the structural analysis of glycoproteins and their glycan moieties. Herein, a panel of 13 novel PNGase H+ candidates (the suffix H+ refers to the acidic pH optimum of these acidobacterial PNGases) was tested in their recombinant form for their deglycosylation performance. One candidate (originating from the bacterial species Dyella japonica) showed superior properties both in solution-phase and immobilized on amino-, epoxy- and nitrilotriacetate resins when compared to currently acidic available PNGases. The high expression yield compared to a previously described PNGase H+, broad substrate specificity, and good storage stability of this novel N-glycanase makes it a valuable tool for the analysis of protein glycosylation.

Probing the conformational impact of detergents on the integral membrane protein LeuT by global HDX-MS.

Neurotransmitter:sodium symporters (NSS) are integral membrane proteins (IMP), responsible for reuptake of neurotransmitters from the synaptic cleft. Due to challenges in production of mammalian NSS in their active form, the prokaryotic hydrophobic amino acid transporter, LeuT, served here as a steadfast model for elucidation of structure-function relationship. As NSS proteins reside within phospholipid bilayer, they require stabilization by artificial membrane systems upon their extraction. Right choice of artificial membrane system is crucial as suboptimal detergent and/or lipids can lead to destabilization or non-native stabilization. Here we study the effect of related detergents, dodecyl maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG), on the conformational dynamics of LeuT by global HDX-MS, in the presence of functionally relevant ligands. We observed that LeuT is more dynamic when solubilized in DDM compared to LMNG. Moreover, LeuT exhibited increased HDX in the presence of K+ compared to Na+, indicating a more dynamic conformation in the presence of K+. Upon addition of leucine, LeuT underwent additional stabilization relative to the Na+-bound state. Finally, peak broadening was observed, suggesting that LeuT undergoes slow unfolding/refolding dynamics in detergent solution. These slow dynamics were verified by local HDX, also proving that detergents modulate the rate of these dynamics. SIGNIFICANCE: Overall, we show the efficacy of global HDX-MS to evaluate the effect of artificial membrane systems on integral membrane proteins and the importance of carefully selecting compatible detergent (and/or lipid) for the solubilization of this class of proteins.

Hydrogen/deuterium exchange mass spectrometry with improved electrochemical reduction enables comprehensive epitope mapping of a therapeutic antibody to the cysteine-knot containing vascular endothelial growth factor.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has become a popular method for analysis of the conformational dynamics and interactions of proteins. Disulfide-bonded proteins, however, present a challenge to HDX-MS as they require efficient disulfide bond reduction prior to enzymatic proteolysis. Electrochemical reduction (ER) provides an attractive solution to tackle disulfide-bonded proteins that are resistant to conventional chemical reduction during HDX-MS. However, ER-enabled HDX-MS has been limited by technical challenges including partial unwanted protein oxidation side-reactions, incompatibility with certain buffer components and most importantly, a lack of overall method robustness. In this study, we have sought to address these challenges. We perform a systematic screening of the compatibility of ER to buffers commonly used in HDX-MS samples by using a reliable and simple system suitability test (SST). Furthermore, we demonstrate the benefits of a new design of the electrochemical cell (EC) for ER-enabled HDX-MS, which include a) high repeatability and robustness over large sample batches without the need for electrode polishing and b) high reduction efficiency of disulfide-bonded proteins without unwanted oxidation side-reactions. We show the real-world applicability of the optimized ER-enabled HDX-MS workflow by performing an epitope mapping of a Fab fragment of a therapeutic monoclonal antibody (mAb) to the cysteine knot-containing vascular endothelial growth factor (VEGF). The results allow us to comprehensively map sites in VEGF involved in mAb binding. Overall, our findings show how ER and HDX-MS can be combined to enable analysis of the conformation and interactions of challenging disulfide-rich proteins.