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Descriptions of materials, metrological methods, computational methods, and supplementary results. Figures of HDX-MS publications and citations versus publication year, histogram of peptide sequence lengths, sequence coverage maps, performance of instrumentsoftware configurations, repeatability plots, %E corrected peptide t HDX versus log 10 (t HDX ) for eight peptides. Tables of instrumentation,software, peptide search methodology, and operating conditions of proteolytic, chromatographic components, and effects of peptide charge on deuterium uptake (PDF)

No abstract

Glycans have an immense number of biological activities, necessitating increased efforts to characterize glycan structures. Mass spectrometry has been coupled to electrospray ionization (ESI) to characterize carbohydrates. While the gas-phase structures of glycan− and carbohydrate−metal adducts have been characterized, several questions persist concerning the mechanism of transfer of carbohydrates from ESI droplets into the gas phase. Using various computational methods, including molecular dynamics, steered molecular dynamics, and density functional theory calculations, we present a mechanistic investigation on the evaporation of solvent from nanosized droplets, formation of carbohydrate−metal adducts, and their subsequent release into the gas phase. We relate the computational results to mass spectra of melezitose, a model carbohydrate, and its permethylated derivative. Our results confirm two mechanisms for the release of carbohydrate−ion adducts from solvated droplets. Native (unmodified) carbohydrates are ionized via the charged residue model, while the permethylated derivative is ionized via the ion evaporation model. For both mechanisms, the monomer carbohydrate−metal adduct is the dominant species observed. This work illustrates that the ionization mechanisms are dictated by interactions between the carbohydrate and solvent, and coordination of the carbohydrate with the metal ion. Thus, these results provide insight into the molecular interactions that govern the mechanism of release.

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.

Characterizing glycans is analytically challenging since glycans are heterogeneous, branched polymers with different three-dimensional conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been used to analyze native conformations and dynamics of biomolecules by measuring the mass increase of analytes as labile protons are replaced with deuterium following exposure to deuterated solvents. The rate of exchange is dependent on the chemical functional group, the presence of hydrogen bonds, pH, temperature, charge, and solvent accessibility. HDX-MS of carbohydrates is challenging due to the rapid exchange rate of hydroxyls. Here, we describe an observed HDX reaction associated with residual solvent vapors saturating electrospray sources. When undeuterated melezitose was infused after infusing DO, samples with up to 73% deuterium exchange were detected. This residual solvent HDX was observed for both carbohydrates and peptides in multiple instruments and dependent on sample infusion rate, infusion time, and deuterium content of the solvent. This residual solvent HDX was observed over several minutes of sample analysis and persisted long enough to alter the measured deuterium labeling and possibly change the interpretation of the results. This work illustrates that residual solvent HDX competes with in-solution HDX for rapidly exchanging functional groups. Thus, we propose conditions to minimize this effect, specifically for top-down, in-electrospray ionization, and quench-flow HDX experiments. Graphical Abstract ᅟ.

Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies.

Microsecond reaction times for in-droplet hydrogen/deuterium exchange of carbohydrate hydroxyls have been varied by changing the opening sizes of theta-electrospray emitters.