ABSTRACT
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.
Subject(s)
Meningococcal Infections , Meningococcal Vaccines , Neisseria meningitidis , Humans , Epitope Mapping/methods , Neisseria meningitidis/metabolism , Deuterium/metabolism , Bacterial Proteins/metabolism , Meningococcal Infections/prevention & control , Carrier Proteins , Deuterium Exchange Measurement , Complement Factor H , Antigens, Bacterial , Epitopes , Antibodies, Monoclonal/metabolism , Hydrogen Deuterium Exchange-Mass SpectrometryABSTRACT
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.
Subject(s)
Glycopeptides , Hydrogen , Glycopeptides/chemistry , Deuterium , Peptides/chemistry , Glycoproteins/chemistry , Polysaccharides/chemistryABSTRACT
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.
Subject(s)
Glycoproteins , Polysaccharides , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Glycoproteins/analysis , Glycosylation , Polysaccharides/metabolismABSTRACT
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.
Subject(s)
Asparagine , Hydrogen Deuterium Exchange-Mass Spectrometry , Amidohydrolases/metabolism , Animals , Deuterium Exchange Measurement , Disulfides , Eukaryota/metabolism , Gammaproteobacteria , Glycoproteins/chemistry , Horseradish Peroxidase/metabolism , Lactoferrin/metabolism , Mice , Oligosaccharides , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Polysaccharides/chemistry , SoilABSTRACT
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.
Subject(s)
Dopamine Plasma Membrane Transport Proteins , Symporters , Animals , Dopamine/metabolism , Dopamine Plasma Membrane Transport Proteins/metabolism , Drosophila/metabolism , Ion Transport , Ions/metabolism , Neurotransmitter Agents/metabolism , Potassium/metabolism , Serotonin Plasma Membrane Transport Proteins/metabolism , Sodium/metabolism , Symporters/metabolismABSTRACT
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.
Subject(s)
Molecular Conformation , Neurotransmitter Agents , Symporters/chemistry , Temperature , Kinetics , Membrane Proteins , Molecular Dynamics Simulation , Pharmaceutical Preparations , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Protein Conformation , SodiumABSTRACT
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.
Subject(s)
Cytosol/chemistry , HSP70 Heat-Shock Proteins/chemistry , Lysophospholipids/metabolism , Lysosomes/chemistry , Monoglycerides/metabolism , Humans , Molecular ConformationABSTRACT
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.
Subject(s)
Glycoproteins , Hydrogen Deuterium Exchange-Mass Spectrometry , Deuterium , Disulfides , Epitope MappingABSTRACT
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.
Subject(s)
Deuterium Exchange Measurement , Hydrogen Deuterium Exchange-Mass Spectrometry , Chromatography, Gel , Deuterium , Mass SpectrometryABSTRACT
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.
Subject(s)
Hydrogen Deuterium Exchange-Mass Spectrometry , Major Histocompatibility Complex , Peptides/metabolism , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/metabolism , Humans , Protein Binding , Protein ConformationABSTRACT
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.
Subject(s)
Adhesins, Bacterial , Anti-Bacterial Agents , Antibodies, Monoclonal , Epitope Mapping/methods , Hydrogen Deuterium Exchange-Mass Spectrometry/methods , Adhesins, Bacterial/chemistry , Adhesins, Bacterial/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Binding Sites, Antibody , Protein Binding , Surface Plasmon Resonance/methodsABSTRACT
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.
Subject(s)
Microfluidics , Spectrometry, Mass, Electrospray Ionization , Peptides , Proteins , Sulfhydryl CompoundsABSTRACT
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.
Subject(s)
Amidohydrolases/chemistry , Glycoproteins/analysis , Hydrogen Deuterium Exchange-Mass Spectrometry/methods , Animals , Glycosylation , Humans , Mice , Models, Molecular , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry , Peptides/analysisABSTRACT
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.
ABSTRACT
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.
Subject(s)
Detergents , Hydrogen Deuterium Exchange-Mass Spectrometry , Animals , Maltose , Membrane Proteins , Molecular ConformationABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/chemistry , Cysteine/chemistry , Electrochemical Techniques , Epitope Mapping , Hydrogen Deuterium Exchange-Mass Spectrometry , Vascular Endothelial Growth Factors/chemistry , Humans , Oxidation-ReductionABSTRACT
As quantitative analysis of biotherapeutics in cerebrospinal fluid (CSF) with LC-MS becomes increasingly widespread, there is a need for method developments towards higher sensitivity. By using artificial CSF (aCSF) in the development phase, the consumption of costly and sparsely available CSF can be limited. The aCSF compositions tested here were made from various dilutions of bovine serum albumin (BSA) or rat plasma to mimic the total protein concentration found in CSF. Focusing on monoclonal antibodies, the aCSF was spiked with human immunoglobulin (hIgG) and prepared with the bottom-up analysis technique using LC-MS. Assuming that the composition of the aCSF would affect the digest, the response from aCSF matrices was compared with CSF from rat, monkey, and dog in terms of estimated sample concentration and matrix effects. The samples were spiked with hIgG in the range of 10 to 1000 ng/mL and volumes of 10 µL were transferred to sample preparation. The results indicate that BSA dilutions from 300 to 2000 µg/mL and rat plasma dilutions of 0.5-2% provide the most accurate concentration estimates when compared with rat CSF. 1000 µg/mL BSA did not produce significantly different concentration estimates for 500 ng/mL samples when compared with CSF from rat, monkey, and dog, and can therefore be used as aCSF for several different species.
Subject(s)
Antibodies/cerebrospinal fluid , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Animals , Antibodies/chemistry , Calibration , Dogs , Haplorhini , Humans , Rats , Reference StandardsABSTRACT
We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.
Subject(s)
Cysteine/chemistry , Enzyme Inhibitors/pharmacology , Monoacylglycerol Lipases/antagonists & inhibitors , Thiazoles/pharmacology , Allosteric Regulation , Animals , Binding Sites , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , HeLa Cells , Humans , Mice , Molecular Docking Simulation , Molecular Structure , Monoacylglycerol Lipases/genetics , Monoacylglycerol Lipases/metabolism , Mutagenesis, Site-Directed , Mutation , Oxidation-Reduction , Protein Binding , Rats , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/metabolismABSTRACT
Insight into the structure-function relationship of membrane proteins is important to understand basic cell function and inform drug development, as these are common targets for drugs. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established technique for the study of protein conformational dynamics and has shown compatibility with membrane proteins. However, the digestion and mass analysis of peptides from membrane proteins can be challenging, severely limiting the HDX-MS experiment. Here we compare the digestion of four integral membrane proteins-Cl-/H+ exchange transporter (ClC-ec1), leucine transporter (LeuT), dopamine transporter (DAT), and serotonin transporter (SERT)-by the use of porcine pepsin and three alternative aspartic proteases either in-solution or immobilized on-column in an optimized HDX-MS-compatible workflow. Pepsin was the most favorable for the digestion of ClC-ec1 and LeuT, providing coverage of 82.2 and 33.2% of the respective protein sequence; however, the alternative proteases surpassed pepsin for the digestion of DAT and SERT. By also screening quench solution additives, we observe that the denaturant urea was beneficial, resulting in improved sequence coverage of all membrane proteins, in contrast to guanidine hydrochloride. Furthermore, significant improvements in sequence coverage were achieved by tailoring the chromatography to handle hydrophobic peptides. Overall, we demonstrate that the susceptibility of membrane proteins to proteolytic digestion during HDX-MS is highly protein-specific. Our results highlight the importance of having multiple proteases and different quench buffer additives in the HDX-MS toolbox and the need to carefully screen a range of digestion conditions to successfully optimize the HDX-MS analysis of integral membrane proteins.
Subject(s)
Antiporters/analysis , Dopamine Plasma Membrane Transport Proteins/analysis , Drosophila Proteins/analysis , Escherichia coli Proteins/analysis , Hydrogen Deuterium Exchange-Mass Spectrometry/methods , Peptide Fragments/analysis , Serotonin Plasma Membrane Transport Proteins/analysis , Amino Acid Sequence , Animals , Antiporters/chemistry , Aquifex , Aspartic Acid Proteases/chemistry , Bacteria , Dopamine Plasma Membrane Transport Proteins/chemistry , Drosophila Proteins/chemistry , Drosophila melanogaster , Escherichia coli , Escherichia coli Proteins/chemistry , Humans , Models, Molecular , Pepsin A/chemistry , Proteolysis , Serotonin Plasma Membrane Transport Proteins/chemistry , Structure-Activity Relationship , Swine , Urea/chemistryABSTRACT
The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.