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Hydrogen exchange mass spectrometry

Ke Sherry Li, Liuqing Shi, Michael L Gross
Assessment of protein structure and interaction is crucial for understanding protein structure/function relationships. Compared to high-resolution structural tools, including X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-EM, and traditional low-resolution methods, such as circular dichroism, UV-vis, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resolution (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure...
February 16, 2018: Accounts of Chemical Research
Jingjie Mo, Renzhe Jin, Qingrong Yan, Izabela Sokolowska, Michael J Lewis, Ping Hu
Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1...
February 13, 2018: MAbs
Shawn Sternisha, Peilu Liu, Alan G Marshall, Brian G Miller
Human glucokinase (GCK) acts as the body's primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in gck produce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and β-type. Steady-state viscosity variation studies indicate that the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value of a β-type variant is viscosity-independent...
February 9, 2018: Biochemistry
Tobias Kromann-Hansen, Eva Louise Lange, Ida K Lund, Gunilla Høyer-Hansen, Peter A Andreasen, Elizabeth A Komives
The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinase-type plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies...
2018: PloS One
Rupesh Bommana, Qing Chai, Christian Schöneich, William F Weiss, Ranajoy Majumdar
This work compares the conformational stability, backbone flexibility, and aggregation propensity of monomer and dimer fractions of an IgG1 monoclonal antibody (mAb) generated upon UVA light exposure for up to 72 hours collected by preparative size-exclusion chromatography (SEC), compared to unstressed control. UVA light exposure induced covalent aggregation, and fragmentation as measured by SEC, sodium dodecyl sulfate polyacrylamide gel electrophoresis and extensive oxidation of specific methionine residues (Met 257, Met 433, and Met 109) in both size fractions identified by reverse phase chromatography coupled to mass spectrometry...
January 30, 2018: Journal of Pharmaceutical Sciences
Gonghua Yang, Yanlong Wei, Zhenzhu Huang, Jiwen Hu, Guojun Liu, Ming Ou, Shudong Lin, Yuanyuan Tu
Reported herein is a novel strategy for the rapid and efficient collection of platinum from Karstedt's catalyst solution. By taking advantage of a ligand-exchange reaction between alkynols and the 1,3-divinyltetramethyldisiloxane ligand (MViMVi) that coordinated with platinum (Pt(0)), the Karstedt's catalyst particles with a size of approximately 2.5 ± 0.7 nm could be reconstructed and assembled into larger particles with a size of 150 ± 35 nm due to the hydrogen bonding between the hydroxyl groups of the alkynol...
January 30, 2018: ACS Applied Materials & Interfaces
Shengdong Pan, Xiaohong Chen, Qian He, Xiaohai Li, Li Wang, Jian Zhou, Micong Jin
A method for the characterization of the metabolite pentachlorophenol hydrogen sulfate (PCP-SO3H) of pentachlorophenol (PCP) in loaches was developed based on ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The loach samples exposed in low concentration of PCP solution were firstly crushed, then extracted by acetonitrile-water solution (70:30, v/v) containing 8% (v/v) triethylamine and purified by mixed anion exchange solid-phase extraction (SPE) cartridges. The chromatographic separation was carried out on a Waters ACQUITY BEH C18 column (100 mm×2...
December 8, 2017: Se Pu, Chinese Journal of Chromatography
Xiaoji Cao, Xue Cai, Weimin Mo
RATIONALE: The comparative study of higher-energy collisional dissociation(HCD) and collision-induced dissociation(CID) mechanisms for protonated cyclic indolyl α-amino esters in quadrupole/orbitrap(Q/Orbitrap) and quadrupole time-of-flight(QTOF) mass spectrometers, respectively, is helpful to study the structures and properties of biologically active indole derivatives using tandem mass spectrometry(MS/MS) technology. METHODS: HCD and CID experiments were carried out using electrospray ionization Q/Orbitrap MS and QTOFMS in positive ion mode, respectively...
January 25, 2018: Rapid Communications in Mass Spectrometry: RCM
M Aiman Mohtar, Lenka Hernychova, Robert O'Neill, Melanie Lawrence, Euan Murray, Borek Vojtesek, Ted R Hupp
AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL)...
January 16, 2018: Molecular & Cellular Proteomics: MCP
Thibault Terencio, Jana Roithová, Stéphane Brandès, Yoann Rousselin, Marie-José Penouilh, Michel Meyer
Iron(III) and uranyl complexes of N-methylacetohydroxamic acid (NMAH) have been investigated by mass spectrometry, infrared multiphoton dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations. A comparison between IRMPD and theoretical IR spectra enabled one to probe the structures for some selected complexes detected in the gas phase. The results show that coordination of Fe3+ and UO22+ by hydroxamic acid is of a very similar nature. Natural bond orbital analysis suggests that bonding in uranyl complexes possesses a slightly stronger ionic character than that in iron complexes...
January 16, 2018: Inorganic Chemistry
Nicholas I Brodie, Romain Huguet, Terry Zhang, Rosa Viner, Vlad Zabrouskov, Jingxi Pan, Evgeniy V Petrotchenko, Christoph H Borchers
Top-down hydrogen-deuterium exchange (HDX) analysis using electron capture or transfer dissociation Fourier transform mass spectrometry is a powerful method for the analysis of secondary structure of proteins in solution. The resolution of the method is a function of the extensive fragmentation of backbone bonds in the proteins. While fragmentation is usually extensive near the N- and C-termini, electron capture (ECD) or electron transfer dissociation (ETD) fragmentation methods sometimes lack good coverage of certain regions of the protein - most often in the middle of the sequence...
January 16, 2018: Analytical Chemistry
Andrea Sinz
Structural mass spectrometry (MS) is gaining increasing importance in deriving valuable three-dimensional structural information on proteins and protein complexes and complements existing techniques, such as NMR spectroscopy and X-ray crystallography. Structural MS unites different MS-based techniques, i.e, hydrogen-deuterium exchange, native MS, ion-mobility MS, protein footprinting, and chemical cross-linking/MS, and allows fundamental questions in structural biology to be addressed. In this article, I will focus on the cross-linking/MS strategy...
January 15, 2018: Angewandte Chemie
B Setner, M Wierzbicka, L Jerzykiewicz, M Lisowski, Z Szewczuk
Recently, we developed a novel non-fragmenting quaternary ammonium ionization tag for the mass spectrometric sensitive sequencing of peptides, based on the N-spiro proline residue (5-azoniaspiro[4.4.]nonyl-carbonyl). Herein, we present an unexpected racemization and the hydrogen-deuterium exchange (HDX) at the α-C atom of the proline derivative under basic aqueous conditions (1% water solution of triethylamine). The deuterium atom, substituted for the α-C atom, does not undergo back-exchange under acidic aqueous conditions which makes the deuterated isotopologue a promising stabile isotope-coded internal standard for quantitative analysis by mass spectrometry...
January 12, 2018: Organic & Biomolecular Chemistry
Michael J Pulkoski-Gross, Meredith L Jenkins, Jean-Philip Truman, Mohamed F Salama, Christopher J Clarke, John E Burke, Yusuf A Hannun, Lina M Obeid
Sphingosine kinase 1 (SK1) is a key enzyme which regulates a multitude of cellular processes including cellular growth, immune cell trafficking, and inflammation via the production of sphingosine-1-phosphate (S1P). To produce S1P, SK1 must access sphingosine directly from membranes. Despite the need for membrane access, the molecular mechanisms underlying SK1s direct membrane interaction remain unclear. Here, we identify a novel positively charged site on SK1 which is responsible for electrostatic interactions with membranes...
January 11, 2018: Journal of Lipid Research
Chimno Ihuoma Nnadi, Meredith L Jenkins, Daniel R Gentile, Leslie A Bateman, Daniel Zaidman, Trent E Balius, Daniel K Nomura, John E Burke, Kevan M Shokat, Nir London
The success of targeted covalent inhibitors in the global pharmaceutical industry has led to a resurgence of covalent drug discovery. However, covalent inhibitor design for flexible binding sites remains a difficult task due to lack of methodological development. Here, we compared covalent docking to empirical electrophile screening, against the highly dynamic target K-RasG12C. While the overall hit-rate of both methods was comparable, we were able to rapidly progress a docking hit to a potent irreversible covalent inhibitor that modifies the inactive, GDP-bound state of K-RasG12C...
January 10, 2018: Journal of Chemical Information and Modeling
Monita Muralidharan, Rajdeep Das, Vijay Bhat, Amit Kumar Mandal
Electrostatic attraction between α and β globin chains hold the subunits together in tetrameric human hemoglobin molecule (α₂β₂). Compared to normal globin chains, the affinity of a mutant chain to its partner globin in genetic variants of hemoglobin might be different. This leads to an unequal abundance of normal and variant hemoglobin in heterozygous sample, even though the rates of synthesis of both normal and variant chains are same. The aforementioned affinities across various globin chains might be assessed by the quantification of different forms of tetramers present in a variant hemoglobin sample...
January 8, 2018: Chembiochem: a European Journal of Chemical Biology
Yoshitomo Hamuro, Stephen J Coales
The practice of HDX-MS remains somewhat difficult, not only for newcomers but also for veterans, despite its increasing popularity. While a typical HDX-MS project starts with a feasibility stage where the experimental conditions are optimized and the peptide map is generated prior to the HDX study stage, the literature usually reports only the HDX study stage. In this protocol, we describe a few considerations for the initial feasibility stage, more specifically, how to optimize quench conditions, how to tackle the carryover issue, and how to apply the pepsin specificity rule...
January 3, 2018: Journal of the American Society for Mass Spectrometry
Xiang Ye, Leland Mayne, Zhong-Yuan Kan, S Walter Englander
We used hydrogen exchange-mass spectrometry (HX MS) and fluorescence to compare the folding of maltose binding protein (MBP) in free solution and in the GroEL/ES cavity. Upon refolding, MBP initially collapses into a dynamic molten globule-like ensemble, then forms an obligatory on-pathway native-like folding intermediate (1.2 seconds) that brings together sequentially remote segments and then folds globally after a long delay (30 seconds). A single valine to glycine mutation imposes a definable folding defect, slows early intermediate formation by 20-fold, and therefore subsequent global folding by approximately twofold...
January 2, 2018: Proceedings of the National Academy of Sciences of the United States of America
Darragh P O'Brien, Dominique Durand, Alexis Voegele, Véronique Hourdel, Marilyne Davi, Julia Chamot-Rooke, Patrice Vachette, Sébastien Brier, Daniel Ladant, Alexandre Chenal
Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding...
December 29, 2017: PLoS Biology
Jamie A Moroco, John Jeff Alvarado, Ryan P Staudt, Haibin Shic, Thomas E Wales, Thomas E Smithgall, John R Engen
The HIV-1 accessory protein Nef controls multiple aspects of the viral life cycle and host immune response, making it an attractive therapeutic target. Previous X-ray crystal structures of Nef in complex with key host cell binding partners have shed light on protein-protein interactions critical to Nef function. Crystal structures of Nef in complex with either the SH3 or tandem SH3-SH2 domains of Src-family kinases reveal distinct dimer conformations of Nef. However, the existence of these Nef dimer complexes in solution has not been established...
December 16, 2017: Journal of Molecular Biology
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