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For hydrogen-deuterium exchange mass spectrometry (HDX-MS) to have an increased role in quality control of biopharmaceuticals, H for D back-exchange occurring during protein analyses should be minimized to promote greater reproducibility. Standard HDX-MS analysis systems that digest proteins and separate peptides at pH 2.7 and 0 °C can lose30% of the deuterium marker within 15 min of sample injection. This report describes the architecture and performance of a dual-enzyme, HDX-MS instrument that conducts liquid chromatography (LC) separations at subzero temperature, thereby reducing back-exchange and supporting longer LC separations with improved chromatographic resolution. LC separations of perdeuterated, fully reduced, iodoacetamide-treated BSA protein digest standard peptides were performed at 0, -10, -20, and -30 °C in ethylene glycol (EG)/H

This document presents hydrogen-deuterium exchange mass spectrometry (HDX-MS) data from measurements of three purified IgG1 glycoform samples, predominantly G0F, G2F, and SAF, in isolation and in complexation with the high-affinity receptor, FcγRIa (CD64). The IgG1 antibody used in this study, aIL8hFc, is a murine-human chimeric IgG1, which inhibits IL-8 binding to human neutrophils.

This tutorial provides mechanical drawings, electrical schematics, parts lists, stereolithography (STL) files for producing three-dimensional (3D)-printed parts, initial graphics exchange specification (IGS) files for automated machining, and instructions necessary for construction of a dual protease column, subzero, liquid chromatography system for hydrogen-deuterium exchange mass spectrometry (HDX-MS). Electro-mechanical schematics for construction of two multi-zone temperature controllers that regulate to ±0.05 oC are also included in this tutorial.

Previous reports present different models for the stabilization of the Fc-FcγRI immune complex. Although accord exists on the importance of L235 in IgG1 and some hydrophobic contacts for complex stabilization, discord exists regarding the existence of stabilizing glycoprotein contacts between glycans of IgG1 and a conserved FG-loop (

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 FcγRIIa (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 FcγRIIa 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 FcγRIIa. 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.

The reported data describe the crystallization, crystal packing, structure determination and twinning of the unliganded Fab (antigen-binding fragment) from the NISTmAb (standard reference material 8671). The raw atomic coordinates are available as Protein Data Bank structure 5K8A and biological aspects are described in the article, (Karageorgos et al., 2017) [1]. Crystal data show that the packing is unique, and show the basis for the crystal's twinned growth. Twinning is a common and often serious problem in protein structure determination by x-ray crystallography [2]. In the present case the twinning is due to a small deviation (about 0.3 nm) from 4-fold symmetry in the primary intermolecular interface. The deviation produces pseudosymmetry, generating slightly different conformations of the protein, and alternating strong and weak forms of key packing interfaces throughout the lattice. keywords: Antibody, NIST, Pseudosymmetry, Standard, Twinning

PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn2+ or Mg2+ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn2+ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg2+ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange–mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg2+, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30–300 s. Submillimolar Mg2+ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192–200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg2+ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg2+ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain.

The spreadsheet file reported herein provides centroid data, descriptive of deuterium uptake, for the FabFragment of NISTmAb (PDB: 5K8A) reference material, as measured by the bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS) method. The protein sample was incubated in deuterium-rich solutions under uniform pH and salt concentrations between 3.6 oC and 25.4 oC for seven intervals ranging over (0 to 14,400) s plus a ∞pseudo s control. The deuterium content of peptic peptide fragments were measured by mass spectrometry. These data were reported by fifteen laboratories, which conducted the measurements using orbitrap and Q-TOF mass spectrometers. The cohort reported ≈ 78,900 centroids for 430 proteolytic peptide sequences of the heavy and light chains of NISTmAb, providing nearly 100 % coverage. In addition, some groups reported ≈ 10,900 centroid measurements for 77 peptide sequences of the Fc fragment. The instrumentation and physical and chemical conditions under which these data were acquired are documented.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A) from fifteen laboratories. Laboratories reported ≈ 89,800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (≈ 78,900 centroids), giving ≈ 100 % coverage, and ≈ 10,900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87 %) exhibited centroid mass laboratory repeatability precisions of 〈 sLab 〉 ≤ (0.15 ± 0.01) Da (1σx ̅ ), and all laboratories achieved 〈 sLab 〉 ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) oC and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Labs ( tHDX ) = (9.0 ± 0.9) % (1σ). A 9 laboratory cohort that immersed samples at THDX = 25 oC exhibited reproducibility of σreproducibility25C cohort ( tHDX ) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.

Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls cholesterol elimination from the brain. This P450 is also a potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified by efavirenz, the anti-HIV medication. Previously, we suggested that pharmaceuticals activate CYP46A1 allosterically through binding to a site on the cytosolic protein surface, which is different from the enzyme active site facing the membrane. Here we identified this allosteric site for efavirenz on CYP46A1 by using a combination of hydrogen-deuterium exchange coupled to MS, computational modeling, site-directed mutagenesis, and analysis of the CYP46A1 crystal structure. We also mapped the binding region for the CYP46A1 redox partner oxidoreductase and found that the allosteric and redox partner binding sites share a common border. On the basis of the data obtained, we propose the mechanism of CYP46A1 allostery and the pathway for the signal transmission from the P450 allosteric site to the active site.

An understanding of how conformational dynamics modulates function and catalysis of human monoacylglycerol lipase (hMGL), an important pharmaceutical target, can facilitate the development of novel ligands with potential therapeutic value. Here, we report the discovery and characterization of an allosteric, regulatory hMGL site comprised of residues Trp-289 and Leu-232 that reside over 18 Å away from the catalytic triad. These residues were identified as critical mediators of long-range communication and as important contributors to the integrity of the hMGL structure. Nonconservative replacements of Trp-289 or Leu-232 triggered concerted motions of structurally distinct regions with a significant conformational shift toward inactive states and dramatic loss in catalytic efficiency of the enzyme. Using a multimethod approach, we show that the dynamically relevant Trp-289 and Leu-232 residues serve as communication hubs within an allosteric protein network that controls signal propagation to the active site, and thus, regulates active-inactive interconversion of hMGL. Our findings provide new insights into the mechanism of allosteric regulation of lipase activity, in general, and may provide alternative drug design possibilities.

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that associates with and influences the activity of many proteins participating in DNA metabolic processes and cell cycle progression. Previously, an uncharacterized small protein, encoded by TK0808 in the archaeon Thermococcus kodakarensis, was shown to stably interact with PCNA in vivo. Here, we show that this protein, designated Thermococcales inhibitor of PCNA (TIP), binds to PCNA in vitro and inhibits PCNA-dependent activities likely by preventing PCNA trimerization. Using hydrogen/deuterium exchange mass spectrometry and site-directed mutagenesis, the interacting regions of PCNA and TIP were identified. Most proteins bind to PCNA via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA. TIP, however, lacks any known PCNA-interacting motif, suggesting a new mechanism for PCNA binding and regulation of PCNA-dependent activities, which may support the development of a new subclass of therapeutic biomolecules for inhibiting PCNA.

Biological processes are the result of noncovalent, protein-ligand interactions, where the ligands range from small organic and inorganic molecules to lipids, nucleic acids, peptides, and proteins. Amide groups within proteins constantly exchange protons with water. When immersed in heavy water (D2O), mass spectrometry (MS) can measure the change of mass associated with the hydrogen to deuterium exchange (HDX). Protein-ligand interactions modify the hydrogen exchange rates of amide protons, and the measurement of the amide exchange rates can provide rich information regarding the dynamical structure of the protein-ligand complex. This chapter describes a protocol for conducting bottom-up, continuous uptake, proteolytic fragmentation HDX-MS experiments that can help identify and map the interacting peptides of a protein-ligand interface. This tutorial outlines the fundamental theory governing hydrogen exchange; provides practical information regarding the preparation of protein samples and solutions; and describes the exchange reaction, reaction quenching, enzymatic digestion, chromatographic separation, and peptide analysis by MS. Tables list representative combinations of fluidic components used by HDX-MS researchers and summarize the available HDX-MS analysis software packages. Additionally, two HDX-MS case studies are used to illustrate protein-ligand interactions involving: (1) a continuous sequence of interacting residues and (2) a set of discontinuously numbered residues, residing spatially near each other.

Biological processes are the result of noncovalent, protein-ligand interactions, where the ligands range from small organic and inorganic molecules to lipids, nucleic acids, peptides, and proteins. Amide groups within proteins constantly exchange protons with water. When immersed in heavy water (D2O), mass spectrometry (MS) can measure the change of mass associated with the hydrogen to deuterium exchange (HDX). Protein-ligand interactions modify the hydrogen exchange rates of amide protons, and the measurement of the amide exchange rates can provide rich information regarding the dynamical structure of the protein-ligand complex. This chapter describes a protocol for conducting bottom-up, continuous uptake, proteolytic fragmentation HDX-MS experiments that can help identify and map the interacting peptides of a protein-ligand interface. This tutorial outlines the fundamental theory governing hydrogen exchange; provides practical information regarding the preparation of protein samples and solutions; and describes the exchange reaction, reaction quenching, enzymatic digestion, chromatographic separation, and peptide analysis by MS. Tables list representative combinations of fluidic components used by HDX-MS researchers and summarize the available HDX-MS analysis software packages. Additionally, two HDX-MS case studies are used to illustrate protein-ligand interactions involving: (1) a continuous sequence of interacting residues and (2) a set of discontinuously numbered residues, residing spatially near each other.

Triphenylphosphine (PPh(3)) is commonly used during syntheses of stable, closed-shell monolayer protected clusters (MPCs). Models of transition metal (TM) cluster and nanoparticle syntheses commonly assign PPh(3) a passive role as a chemical placeholder, electron balancing species, or surfactant. This study provides the first direct evidence that PPh(3) is a proactive etching agent that promotes the formation of specific closed-shell cluster sizes. To observe this effect, we developed a colorimetric tool that simultaneously monitors size distribution and population of PPh(3)-protected clusters as a function of time. The distribution of the clusters is assigned to different bin sizes by chemical conversion with L(3) (L(3) = 1,3-bis(diphenylphosphino)propane): (i) total conversion of PPh(3)-protected Au(8) and Au(9) clusters into [Au(6)L(3)(4)](2+) and (ii) ligand exchange with [Au(x)(PPh(3))(y)](z+) (10 ≤ x ≤ 13) clusters to form L(3)-protected Au(10) and Au(11) clusters. Evolution of the nascent cluster distribution in ethanol and methanol solvent systems was monitored by the colorimetric assay, which revealed a cyclic process of growth and etching reactions around the most stable cluster species to form nearly monodisperse product distributions. We formally define the population growth of specific clusters through cyclic processing of the Au MPCs as "size selective" processing. The current study highlights the need for incorporating bidirectional processing, including relative rate information, into TM kinetic models for ligands with growth and etching efficacy.

We report a new strategy that provides stringent control for the size and dispersity of ultrasmall nanoclusters through preparation of gold complex distributions formed from the precursor, AuClPPh3 (PPh3 = triphenylphosphine), and the L6 (L6 = 1,6-bis(diphenylphosphino) hexane) ligand prior to reduction with NaBH4 in 1:1 methanol/chloroform solutions. Monodisperse nanoclusters of distinct nuclearity are obtained for specific ligand ratios; [L6]/[PPh3] = 4 yields [Au8L64]2+, [L6]/[PPh3] = 0.4 yields [Au9L64Cl]2+, and [L6]/[PPh3] = 8 yields ligated Au10 cores in the form of [Au10L64]2+ and [Au10L65]2+. Polyhedral skeletal electron pair theory accounts for the stability of [Au9L64Cl]2+, which is the smallest closed-shell chlorinated cluster reported. Electrospray mass spectrometry and UV−vis spectra indicate that [Au9L64Cl]2+ and [Au10L6x]2+ (x = 4, 5) result from reactions involving [Au8L64]2+. Syntheses of small gold clusters containing chloride ligands open the possibility of constructing larger clusters ...

Ligand exchange reactions important in the formation of L3- and L5-protected gold clusters (L3 = 1,3-bis(diphenylphosphino)propane; L5 = 1,5-bis(diphenylphosphino)pentane) are investigated at the molecular level. We establish that molecular complexes, [Au(PPh3)xL2-xn]+, in which Au+ is bound to at least one triphenylphosphine ligand (PPh3) act as precursors to cluster formation, while complexes in which Au+ is bound only to the diphosphine ligands do not. Ligand exchange reactions and ion equilibria were studied via electrospray ionization mass spectrometry, and a critical reaction was studied with ab initio molecular dynamics simulations. The displacement of one PPh3 by L3 on [Au(PPh3)2]+ was studied with transition path sampling calculations with Car−Parrinello molecular dynamics simulations. The experiments and simulations are in excellent agreement, and we demonstrate the applicability of these tools to chemical reactions involving ligand exchange.

Car-Parrinello molecular dynamics (CPMD) simulations, DFT chemical reactivity index calculations, and mass spectrometric measurements are combined in an integrated effort to elucidate the details of the coordination of a transition-metal ion to a carbohydrate. The impact of the interaction with the FeIII ion on the glycosidic linkage conformation of methyl-alpha-d-mannopyranoside is studied by classical molecular dynamics (MD) and CPMD simulations. This study shows that FeIII interacts with specific hydroxyl oxygen atoms of the carbohydrate, affecting the ground state carbohydrate conformation. These conformational details are discussed in terms of a set of supporting experiments involving electrospray ionization mass spectrometry, and CPMD simulations clearly indicate that the specific conformational preference is due to intramolecular hydrogen bonding. Classical MD simulations proved insensitive to these important chemical properties. Thus, we demonstrate the importance of chemical reactivity calculations and CPMD simulations in predicting the active sites of biological molecules toward metal cations.

Highly monodisperse samples of diphosphine ligand (1,3-bis(diphenylphosphino)propane- or 1,5-bis(diphenylphosphino)pentane)-protected gold nanoparticles form rapidly in a mixed methanol/chloroform solvent environment. Methanol soluble octagold, decagold, and undecagold monolayer protected clusters yield very stable ion currents when introduced into a mass spectrometer via electrospray ionization. In addition to neutral ligand loss pathways, collision-induced dissociation generates [AuL]+ and [Au3L]+ (L = diphosphine ligand) as particularly stable product ions from all clusters considered. Furthermore, deca- and undecagold clusters are found to be more resistant to collision induced dissociation, and more susceptible to partial ligand losses than octagold clusters. This suggests that for the deca- and undecagold species, Au−P and P−Ph (Ph = phenyl) bonds within the ligand-protected clusters are of comparable strength.