Mechanistic target of rapamycin (mTOR) is a key protein kinase that integrates various internal and external signals to control biological events including cell growth. Whereas substantial efforts were made to elucidate protein subunits interacting with mTOR, endogenous metabolite-mTOR interactions remain largely unknown. Using affinity protein purification and mass spectrometry, we identified direct binding of mTOR to 13- S -hydroxyoctadecadienoic acid (13- S -HODE) which is an oxygenated metabolite of linoleic acid, a polyunsaturated essential fatty acid. Interaction of 13- S -HODE with the catalytic ATP-binding domain of mTOR prevented its kinase activity in an ATP-competitive manner. Furthermore, either 13- S -HODE treatment or expression of arachidonate 15-lipoxygenase (ALOX15), an enzyme responsible for 13- S -HODE production, reduced mTOR signaling, thereby suppressing the growth of cancer cells as well as tumor xenografts. Our results highlight the importance of 13- S -HODE serving as a tumor suppressive, mTOR-inhibiting metabolite that links polyunsaturated fatty acid metabolism and the mTOR signaling in controlling cancer cell growth. ### Competing Interest Statement The authors have declared no competing interest.

Antibodies are thought to play a major role in protection against human norovirus infection. Mouse humoral responses closely mimic those of humans; thus, mouse models are used to characterize norovirus epitopes on the major viral capsid protein, VP1. We have developed a panel of mouse monoclonal antibodies (mAbs) produced against the last pandemic variant to emerge, Sydney 2012. While most mAbs (25/44) were mapped to variable antigenic sites on VP1, 19 of the mAbs were cross-reactive against multiple genotypes or GII.4 variants. Most (12/19) of the cross-reactive mAbs bound to the Shell domain and were cross-reactive with different GII noroviruses. Interestingly, mAb 30A11 exhibited cross-reactivity against all tested norovirus genotypes (GI, GII, GIV, and GIX). This mAb was mapped to a highly conserved region of the Shell domain (51PIDPWII57) using peptide ELISA and immunofluorescence. Of those mapping to the Protruding (P) domain, two (19C10 and 14B11) showed cross-reactivity with GII noroviruses. Using hydrogen-deuterium exchange mass spectrometry, we mapped 19C10 to a conserved region of the P domain near the P/Shell interface, which explains its cross-reactivity with different GII noroviruses and lack of histo-blood group antigen-blocking activity. Binding and mutational analyses showed that residues 518, 519, and 525 are important for 19C10 and 14B11 epitope recognition. While the antibodies described here are mostly non-neutralizing, they can be useful tools for research and diagnostics of noroviruses. The role of non-neutralizing, cross-reactive antibodies targeting different areas of the viral capsid merits further research to facilitate our understanding of immunity to norovirus infection and disease. IMPORTANCE:To gain insights into the overall immune responses to human norovirus, we characterized non-neutralizing, cross-reactive monoclonal antibodies (mAbs) developed against a pandemic GII.4 norovirus. We determined the binding epitope of an antibody that exhibited cross-reactivity against all tested noroviruses, which makes it a useful tool for research and diagnostics. The epitope of two additional non-neutralizing mAbs was mapped to a less conserved region on the viral capsid protein, explaining their cross-reactivity patterns. Often overlooked, the role of non-neutralizing, cross-reactive mAbs merits further research to facilitate our understanding of immunity to norovirus infection and disease.

ABSTRACT Many multi-domain proteins including the serpin family of serine protease inhibitors contain non-sequential domains composed of regions that are far apart in sequence. Because proteins are translated vectorially from N-to C-terminus, such domains pose a particular challenge: how to balance the conformational lability necessary to form productive interactions between early and late translated regions while avoiding aggregation. This balance is mediated by the protein sequence properties and the interactions of the folding protein with the cellular quality control machinery. For serpins, particularly α 1 -antitrypsin (AAT), mutations often lead to polymer accumulation in cells and consequent disease suggesting that the lability/aggregation balance is especially precarious. Therefore, we investigated the properties of progressively longer AAT N-terminal fragments in solution and in cells. The N-terminal subdomain, residues 1-190 (AAT190), is monomeric in solution and efficiently degraded in cells. More ý-rich fragments, 1-290 and 1-323, form small oligomers in solution, but are still efficiently degraded, and even the polymerization promoting Siiyama (S53F) mutation did not significantly affect fragment degradation. In vitro, the AAT190 region is among the last regions incorporated into the final structure. Hydrogen-deuterium exchange mass spectrometry and enhanced sampling molecular dynamics simulations show that AAT190 has a broad, dynamic conformational ensemble that helps protect one particularly aggregation prone ý-strand from solvent. These AAT190 dynamics result in transient exposure of sequences that are buried in folded, full-length AAT, which may provide important recognition sites for the cellular quality control machinery and facilitate degradation and, under favorable conditions, reduce the likelihood of polymerization.

Hydrogen-deuterium exchange (HDX) is a comprehensive yet detailed probe of protein structure and dynamics and, coupled to mass spectrometry, has become a powerful tool for investigating an increasingly large array of systems. Computer simulations are often used to help rationalize experimental observations of exchange, but interpretations have frequently been limited to simple, subjective correlations between microscopic dynamical fluctuations and the observed macroscopic exchange behavior. With this in mind, we previously developed the HDX ensemble reweighting approach and associated software, HDXer, to aid the objective interpretation of HDX data using molecular simulations. HDXer has two main functions; first, to compute H-D exchange rates that describe each structure in a candidate ensemble of protein structures, for example from molecular simulations, and second, to objectively reweight the conformational populations present in a candidate ensemble to conform to experimental exchange data. In this article, we first describe the HDXer approach, theory, and implementation. We then guide users through a suite of tutorials that demonstrate the practical aspects of preparing experimental data, computing HDX levels from molecular simulations, and performing ensemble reweighting analyses. Finally we provide a practical discussion of the capabilities and limitations of the HDXer methods including recommendations for a user's own analyses. Overall, this article is intended to provide an up-to-date, pedagogical counterpart to the software, which is freely available at https://github.com/Lucy-Forrest-Lab/HDXer.

The serpin plasminogen activator inhibitor 1 (PAI-1) spontaneously undergoes a massive structural change from a metastable and active conformation, with a solvent-accessible reactive center loop (RCL), to a stable, inactive, or latent conformation, with the RCL inserted into the central β-sheet. Physiologically, conversion to the latent state is regulated by the binding of vitronectin, which hinders the latency transition rate approximately twofold. The molecular mechanisms leading to this rate change are unclear. Here, we investigated the effects of vitronectin on the PAI-1 latency transition using all-atom path sampling simulations in explicit solvent. In simulated latency transitions of free PAI-1, the RCL is quite mobile as is the gate, the region that impedes RCL access to the central β-sheet. This mobility allows the formation of a transient salt bridge that facilitates the transition; this finding rationalizes existing mutagenesis results. Vitronectin binding reduces RCL and gate mobility by allosterically rigidifying structural elements over 40 Å away from the binding site, thus blocking transition to the latent conformation. The effects of vitronectin are propagated by a network of dynamically correlated residues including a number of conserved sites that were previously identified as important for PAI-1 stability. Simulations also revealed a transient pocket populated only in the vitronectin-bound state, corresponding to a cryptic drug-binding site identified by crystallography. Overall, these results shed new light on PAI-1 latency transition regulation by vitronectin and illustrate the potential of path sampling simulations for understanding functional protein conformational changes and for facilitating drug discovery.

BACKGROUND:Deficiency in blood coagulation factor VIII (FVIII) results in life-threating bleeding (hemophilia A) treated by infusions of FVIII concentrates. To improve disease treatment, FVIII has been modified to increase its plasma half-life, which requires understanding mechanisms of FVIII catabolism. An important catabolic actor is hepatic low density lipoprotein receptor-related protein 1 (LRP1), which also regulates many other clinically significant processes. Previous studies showed complexity of FVIII site for binding LRP1.OBJECTIVES:To characterize binding sites between FVIII and LRP1 and suggest a model of the interaction.METHODS:A series of recombinant ligand-binding complement-type repeat (CR) fragments of LRP1 including mutated variants was generated in a baculovirus system and tested for FVIII interaction using surface plasmon resonance, tissue culture model, hydrogen-deuterium exchange mass spectrometry, and in silico.RESULTS:Multiple CR doublets within LRP1 clusters II and IV were identified as alternative FVIII-binding sites. These interactions follow the canonical binding mode providing major binding energy, and additional weak interactions are contributed by adjacent CR domains. A representative CR doublet was shown to have multiple contact sites on FVIII.CONCLUSIONS:FVIII and LRP1 interact via formation of multiple complex contacts involving both canonical and non-canonical binding combinations. We propose that FVIII-LRP1 interaction occurs via switching such alternative binding combinations in a dynamic mode, and that this mechanism is relevant to other ligand interactions of the low-density lipoprotein receptor family members including LRP1.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a valuable approach to probe protein structure and dynamics. However, interpretation of HDX-MS data is typically limited to peptide-level resolution. Ongoing efforts aim to integrate experimental HDX-MS data with molecular dynamics (MD) approaches, providing a high resolution interpretation of the HDX-MS data. Thus, an HDX-MS based maximum-entropy reweighting approach (HDXer) was developed to reweight computationally generated ensembles to HDX-MS data.

Probing the structural equilibrium that proteins and protein complexes adopt in solution is critical in understanding a multitude of its biophysical properties. Two common ways to probe these dynamic structures are hydrogen deuterium exchange coupled mass spectrometry (HDX-MS) and molecular dynamics simulations (MD). HDX-MS is a solution-based technique that reports on a system's structure and dynamics in solution at peptide level resolution. Although informative, the typical information obtained through HDX-MS studies remains largely qualitative and is limited by the experimentally attainable resolution. On the other hand, MD takes a starting high-resolution structure with a set of model parameters to simulate the system's motion over time. The resulting trajectory gives an atomistic view of the system. However, the time scales accessible to MD simulations are limited and can lead to under exploration of the conformational landscape. Enhanced computational sampling methods have been developed to more efficiently explore a system's conformational landscape. However, determining the biological relevance of these enhanced sampling methods remains difficult. Here we apply a workflow which leverages experimental HDX-MS data, through maximum entropy reweighing, to extract representative ensembles from enhanced MD simulations. These extracted ensembles yield an atomistic resolution model of a protein's/system's in-solution dynamics. We further demonstrate the functionality of this workflow in probing biophysical phenomena. Thus far we have shown the ability to use this workflow to extract representative ensembles of a proteins in-solution native state ensemble (PhuS), protein ligand binding (GAPDH), and identifying metastable protein folding intermediates (A1AT). These high-resolution models are then orthogonally verified through various other biophysical methods. The resulting ensembles are then used to generate further structure-function hypotheses or to begin computer aided drug design

Lon is an ATP-dependent protease belonging to the "ATPase associated with diverse cellular activities" (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The Km values for the intrinsic as well as protein-stimulated ATPase were increased whereas the kcat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings.

The cytoplasmic heme binding protein from Pseudomonas aeruginosa, PhuS, plays two essential roles in regulating heme uptake and iron homeostasis. First, PhuS shuttles exogenous heme to heme oxygenase (HemO) for degradation and iron release. Second, PhuS binds DNA and modulates the transcription of the prrF/H small RNAs (sRNAs) involved in the iron-sparing response. Heme binding to PhuS regulates this dual function, as the unliganded form binds DNA, whereas the heme-bound form binds HemO. Crystallographic studies revealed nearly identical structures for apo- and holo-PhuS, and yet numerous solution-based measurements indicate that heme binding is accompanied by large conformational rearrangements. In particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of apo- versus holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7, and 8 (α6,7,8), which contribute to the heme binding pocket. These helices were mostly labile in apo-PhuS but largely protected in holo-PhuS. In contrast, in silico-predicted deuterium uptake levels of α6,7,8 from molecular dynamics (MD) simulations of the apo- and holo-PhuS structures are highly similar, consistent only with the holo-PhuS HDX-MS data. To rationalize this discrepancy between crystal structures, simulations, and observed HDX-MS, we exploit a recently developed computational approach (HDXer) that fits the relative weights of conformational populations within an ensemble of structures to conform to a target set of HDX-MS data. Here, a combination of enhanced sampling MD, HDXer, and dimensionality reduction analysis reveals an apo-PhuS conformational landscape in which α6, 7, and 8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 toward the HemO binding interface. Circular dichroism analysis confirms the loss of secondary structure, and the extracted ensembles of apo-PhuS and of heme-transfer-impaired H212R mutant, are consistent with known heme binding and transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.

S-nitroso-l-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase–independent (sGC-independent) effects are stereoselective — that is, not recapitulated by S-nitroso-d-cysteine (D-CSNO) — and are inhibited by chemical congeners. However, candidate L-CSNO receptors have not been identified. Here, we have used 2 complementary affinity chromatography assays — followed by unbiased proteomic analysis — to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO–Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy; hydrogen deuterium exchange; and, in Kv1.1/Kv1.2/Kvβ2-overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv1.1 prevented this effect. In intact animals, L-CSNO injection at the level of the carotid body dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO congener S-methyl-l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.

Fucosylation is important for the function of many proteins with biotechnical and medical applications. Alpha-fucosidases comprise a large enzyme family that recognizes fucosylated substrates with diverse α-linkages on these proteins. Lactobacillus casei produces an α-fucosidase, called AlfC, with specificity towards α(1,6)-fucose, the only linkage found in human N- glycan core fucosylation. AlfC and certain point mutants thereof have been used to add and remove fucose from monoclonal antibody N- glycans, with significant impacts on their effector functions. Despite the potential uses for AlfC, little is known about its mechanism. Here, we present crystal structures of AlfC, combined with mutational and kinetic analyses, hydrogen–deuterium exchange mass spectrometry, molecular dynamic simulations, and transfucosylation experiments to define the molecular mechanisms of the activities of AlfC and its transfucosidase mutants. Our results indicate that AlfC creates an aromatic subsite adjacent to the active site that specifically accommodates GlcNAc in α(1,6)-linkages, suggest that enzymatic activity is controlled by distinct open and closed conformations of an active-site loop, with certain mutations shifting the equilibrium towards open conformations to promote transfucosylation over hydrolysis, and provide a potentially generalizable framework for the rational creation of AlfC transfucosidase mutants.

Computational simulations of protein folding can be used to interpret experimental folding results, to design new folding experiments, and to test the effects of mutations and small molecules on folding. However, whereas major experimental and computational progress has been made in understanding how small proteins fold, research on larger, multidomain proteins, which comprise the majority of proteins, is less advanced. Specifically, large proteins often fold via long-lived partially folded intermediates, whose structures, potentially toxic oligomerization, and interactions with cellular chaperones remain poorly understood. Molecular dynamics based folding simulations that rely on knowledge of the native structure can provide critical, detailed information on folding free energy landscapes, intermediates, and pathways. Further, increases in computational power and methodological advances have made folding simulations of large proteins practical and valuable. Here, using serpins that inhibit proteases as an example, we review native-centric methods for simulating the folding of large proteins. These synergistic approaches range from Gō and related structure-based models that can predict the effects of the native structure on folding to all-atom-based methods that include side-chain chemistry and can predict how disease-associated mutations may impact folding. The application of these computational approaches to serpins and other large proteins highlights the successes and limitations of current computational methods and underscores how computational results can be used to inform experiments. These powerful simulation approaches in combination with experiments can provide unique insights into how large proteins fold and misfold, expanding our ability to predict and manipulate protein folding.

Antibody structure couples adaptive and innate immunity via Fab (antigen binding) and Fc (effector) domains that are connected by unique hinge regions. Because antibodies harbor two or more Fab domains, they are capable of crosslinking multi-determinant antigens, which is required for Fc-dependent functions through associative interactions with effector ligands, including C1q and cell surface Fc receptors. The modular nature of antibodies, with distal ligand binding sites for antigen and Fc-ligands, is reminiscent of allosteric proteins, suggesting that allosteric interactions might contribute to Fc-mediated effector functions. This hypothesis has been pursued for over 40 years and remains unresolved. Here, we provide evidence that allosteric interactions between Fab and Fc triggered by antigen binding modulate binding of Fc to low-affinity Fc receptors (FcγR) for a human IgG1. This work opens the path to further dissection of the relative roles of allosteric and associative interactions in Fc-mediated effector functions.

The conformational transition of a fluorinated amphiphilic dendrimer is monitored by the 1 H signal from water, alongside the 19 F signal from the dendrimer. High-field NMR data (chemical shift δ, self-diffusion coefficient D, longitudinal relaxation rate R1 , and transverse relaxation rate R2 ) for both dendrimer (19 F) and water (1 H) match each other in detecting the conformational transition. Among all parameters for both nuclei, the water proton transverse-relaxation rate R2 (1 H2 O) displays the highest relative scale of change upon conformational transition of the dendrimer. Hydrogen/deuterium-exchange mass spectrometry reveals that the compact form of the dendrimer has slower proton exchange with water than the extended form. This result suggests that the sensitivity of R2 (1 H2 O) toward dendrimer conformation originates, at least partially, from the difference in proton exchange efficiency between different dendrimer conformations. Finally, we also demonstrated that this conformational transition could be conveniently monitored using a low-field benchtop NMR spectrometer via R2 (1 H2 O). The 1 H2 O signal thus offers a simple way to monitor structural changes of macromolecules using benchtop time-domain NMR.

Department of Pharmaceutical Sciences, Sch 20 Penn Street, Baltimore, MD 21201, pwintrod@rx.umaryland.edu; Fax: +1 410-7 706-6639 School of Pharmaceutical Sciences, Wuhan † Electronic supplementary information ( relaxation rates vs. concentration of uo and a-cyclodextrin; mass-spectra of concentrations. See DOI: 10.1039/c8ra087 ‡ Current address: Pzer, Inc., Groton, CT Cite this: RSC Adv., 2019, 9, 1956

Febrile-range hyperthermia worsens and hypothermia mitigates lung injury, and temperature dependence of lung injury is blunted by inhibitors of p38 mitogen-activated protein kinase (MAPK). Of the two predominant p38 isoforms, p38α is proinflammatory and p38β is cytoprotective. Here, we analyzed the temperature dependence of p38 MAPK activation, substrate interaction, and tertiary structure. Incubating HeLa cells at 39.5 °C stimulated modest p38 activation, but did not alter tumor necrosis factor-α (TNFα)-induced p38 activation. In in vitro kinase assays containing activated p38α and MAPK-activated kinase-2 (MK2), MK2 phosphorylation was 14.5-fold greater at 39.5 °C than at 33 °C. By comparison, we observed only 3.1- and 1.9-fold differences for activating transcription factor-2 (ATF2) and signal transducer and activator of transcription-1α (STAT1α) and a 7.7-fold difference for p38β phosphorylation of MK2. The temperature dependence of p38α:substrate binding affinity, as measured by surface plasmon resonance, paralleled substrate phosphorylation. Hydrogen-deuterium exchange MS (HDX-MS) of p38α performed at 33, 37, and 39.5 °C indicated temperature-dependent conformational changes in an α helix near the common docking and glutamate:aspartate substrate-binding domains at the known binding site for MK2. In contrast, HDX-MS analysis of p38β did not detect significant temperature-dependent conformational changes in this region. We observed no conformational changes in the catalytic domain of either isoform and no corresponding temperature dependence in the C-terminal p38α-interacting region of MK2. Because MK2 participates in the pathogenesis of lung injury, the observed changes in the structure and function of proinflammatory p38α may contribute to the temperature dependence of acute lung injury.

Immunoglobulin G (IgG) glycosylation critically modulates antibody effector functions. Streptococcus pyogenes secretes a unique endo-β-N-acetylglucosaminidase, EndoS2, which deglycosylates the conserved N-linked glycan at Asn297 on IgG Fc to eliminate its effector functions and evade the immune system. EndoS2 and specific point mutants have been used to chemoenzymatically synthesize antibodies with customizable glycosylation for gain of functions. EndoS2 is useful in these schemes because it accommodates a broad range of N-glycans, including high-mannose, complex, and hybrid types; however, its mechanism of substrate recognition is poorly understood. We present crystal structures of EndoS2 alone and bound to complex and high-mannose glycans; the broad N-glycan specificity is governed by critical loops that shape the binding site of EndoS2. Furthermore, hydrolytic experiments, domain-swap chimeras, and hydrogen–deuterium exchange mass spectrometry reveal the importance of the carbohydrate-binding module in the mechanism of IgG recognition by EndoS2, providing insights into engineering enzymes to catalyze customizable glycosylation reactions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous protein primarily known for its glycolytic function. More recent studies have revealed GAPDH as a multifaceted, multifunctional protein involved in various cellular processes including iron homeostasis, heme transport, apoptosis, tumerogenesis, oxidative stress response, chromatin structure among many others. As part of its multifunctional purpose, GAPDH has been shown to bind to various cellular RNAs and has been classified as a non-canonical RNA binding protein. In particular, it has been shown to bind AU-rich element of mRNA transcripts and consequently affecting translation. However, the detailed structural characterization of the RNA bound complex has remained elusive. The RNA binding site remains unknown thereby restricting potential therapeutic strategies. Our previous studies established that GAPDH binds the AU rich element of TNF-α mRNA transcript via a two-step model. In addition, we have shown that a T229K mutant of GAPDH displayed altered RNA and NAD+ binding properties, potentially signaling to a linkage between RNA binding and the cofactor. Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) revealed that the T229K mutant induced conformational changes into distal grooves along the P-axis of the tetrameric protein. In this study, we have mapped the binding interfaces of three GAPDH constructs displaying varying NAD+ binding properties with the AU rich element from TNF-α mRNA using HDX-MS. A largely common pattern of localized protection from exchange pointed to the grooves along the P-axis as the RNA binding interfaces, as hypothesized. Additionally, some variations in HDX protection point to potential conformational basis for the linkage with NAD+ binding.

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.