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.

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).

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 ) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of 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).

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-β-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.

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.

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.

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-(-acetyl-β-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.

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.

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.

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.

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.

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-1) to investigate the causality between increased binding affinity and conformational dynamics in TCR-pMHC complexes.

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 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.

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.

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.

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

Peptide- -(-acetyl-β-glucosaminyl) asparagine amidases (PNGases, -glycanases, EC 3.5.1.

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 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.

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.

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.

An intact C-terminal end of albumin is required for its long half-life in humans.

Albumin has an average plasma half-life of three weeks and is thus an attractive carrier to improve the pharmacokinetics of fused therapeutics. The half-life is regulated by FcRn, a cellular receptor that protects against intracellular degradation. To tailor-design the therapeutic use of albumin, it is crucial to understand how structural alterations in albumin affect FcRn binding and transport properties.

Investigating surrogate cerebrospinal fluid matrix compositions for use in quantitative LC-MS analysis of therapeutic antibodies in the cerebrospinal fluid.

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.

Molecular Basis of the Mechanisms Controlling MASTL.

The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation.

Benzisothiazolinone Derivatives as Potent Allosteric Monoacylglycerol Lipase Inhibitors That Functionally Mimic Sulfenylation of Regulatory Cysteines.

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--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.

Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.

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.