A5026266291- Mass Spectrometry Techniques and Applications
- Protein purification and stability
- Analytical Chemistry and Chromatography
- Monoclonal and Polyclonal Antibodies Research
- Protein Structure and Dynamics
- Toxin Mechanisms and Immunotoxins
- Enzyme Structure and Function
- Transgenic Plants and Applications
- Atmospheric chemistry and aerosols
- Metabolomics and Mass Spectrometry Studies
- Vector-borne infectious diseases
- Glycosylation and Glycoproteins Research
- Atmospheric Ozone and Climate
- Microfluidic and Capillary Electrophoresis Applications
- Atmospheric aerosols and clouds
- Viral Infections and Vectors
- Advanced Proteomics Techniques and Applications
- Spectroscopy and Laser Applications
- Analytical chemistry methods development
- Receptor Mechanisms and Signaling
- Cytomegalovirus and herpesvirus research
- Signaling Pathways in Disease
- RNA and protein synthesis mechanisms
- Viral Infectious Diseases and Gene Expression in Insects
- Herpesvirus Infections and Treatments
University of Kansas
2016-2025
University of Maryland, Baltimore
2017
University of Rostock
2013
University of New Mexico
2006
Skidmore College
2005
Indiana University Bloomington
1996-1999
Middlebury College
1999
An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability detect the presence multiple conformational states a protein, which often manifest as multimodal isotopic envelopes. However, statistical considerations for accurate analysis spectra have yet be established. Here we outline an unrestrained binomial distribution fitting approach with corresponding tests accurately and, when possible, deconvolute distributions that contain subpopulations....
A Microsoft Excel utility, HX-Express, that significantly accelerates the analysis of hydrogen exchange mass spectrometry data is described. HX-Express generates deuterium uptake and peak width plots from peaks in spectral data. Data intentionally semi-automated, requiring user find to be analyzed. The are entered form x, y lists m/z versus intensity or can directly imported Waters MassLynx software. Analysis with provides same results as manual processing but substantially faster. In...
Proteins that undergo cooperative unfolding events display EX1 kinetic signatures in hydrogen exchange mass spectra. The hallmark bimodal isotope pattern observed for kinetics is distinct from the binomial uncorrelated (EX2), normal regime folded proteins. Detection and characterization of simple when unit large enough isotopic envelopes are resolved m/z scale but become complicated cases where small or there a mixture EX2 kinetics. Here we describe data interpretation method involving peak...
Noisy and overlapped mass spectrometry data hinder the sequence coverage that can be obtained from hydrogen deuterium exchange analysis, places a limit on complexity of samples studied by this technique. Advances in instrumentation have addressed these limits, but as biological under investigation increases, problems are re-encountered. Here we describe use binomial distribution fitting with asymmetric linear squares regression for calculating accurate content envelopes low signal or contain...
This work examines the effect of three anions from Hofmeister series (sulfate, chloride, and thiocyanate) on conformational stability aggregation rate an IgG1 monoclonal antibody (mAb) corresponding changes in mAb's backbone flexibility (at pH 6 25 °C). Compared to a 0.1 M NaCl control, thiocyanate (0.5 M) decreased melting temperatures (Tm) for observed transitions within mAb by 6-9 °C, as measured differential scanning calorimetry. Thiocyanate also accelerated monomer loss at 40 °C over 12...
Differential hydrogen exchange-mass spectrometry (HX-MS) measurements are valuable for identification of differences in the higher order structures proteins. Typically, data sets large with many differential HX values corresponding to peptides monitored at several labeling times. To eliminate subjectivity and reliably identify significant HX-MS measurements, a statistical analysis approach is needed. In this work, we performed null (i.e., no meaningful differences) on maltose binding protein...
Sodium chloride droplets with a median diameter of ∼0.4 μm were generated in the laboratory by atomizing an aqueous solution NaCl under ambient conditions. Infrared extinction spectra aerosols controlled relative humidity (RH) ranging from 15 to 95% obtained. The contained both scattering and absorption components. In order obtain spectrum condensed phase H 2 O associated particulates, it was necessary subtract vapor particulates. subtraction accomplished standard technique. A procedure...
Mapping the structured and disordered regions identifying disorder-to-order transitions are essential to understanding intrinsically proteins (IDPs). One technique that can provide such information is H/D exchange coupled with mass spectrometry (H/D-MS). To explore feasibility of H/D-MS for mapping ordered in IDPs, we undertook a systematic evaluation an unstructured protein, molten globular well-folded complex two proteins. Most segments ACTR (activator thyroid retinoid receptors,...
Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports involvement crystallizable (Fc). In this report, variety biophysical tools, including hydrogen exchange mass spectrometry, are used elucidate protein interface such non-covalent protein-protein...
Measurement of residual structure in intrinsically disordered proteins can provide insights into the mechanisms by which such undergo coupled binding and folding. The present work describes an approach to measure using millisecond hydrogen/deuterium (H/D) exchange a conventional bottom-up peptide-based workflow. We used mid-point, relative totally deuterated control, quantify rate H/D each peptide. A weighted residue-by-residue average these midpoints was map extent at near single-residue...
There is a need for new analytical approaches to better characterize the nature of concentration-dependent, reversible self-association (RSA) monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In work reported here, hydrogen exchange mass spectrometry (HX-MS) was used define concentration-dependent RSA interface, effects association on backbone dynamics an IgG1 mAb (mAb-C). Dynamic light scattering, chemical...
This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein differential scanning calorimetry (DSC) size exclusion chromatography (SEC). The mutation induced differences H/D kinetics at both pH 6.0 7.4. Segments covering sites FcRn binding epitopes...
Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin online digestion an IgG1 monoclonal antibody using immobilized pepsin column under quenched conditions (pH 2.5, 0 °C). From a consensus list 169 different peptides consistently detected during large, ~150 kDa protein, approximately 30% peptic exhibited carry-over. The majority originates from digestion. Carry-over be substantially decreased...
Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein–ligand interactions, protein folding, and dynamics. However, HDX-MS still emergent quality control of biopharmaceuticals establishing dynamic similarity between a biosimilar innovator therapeutic. Because industry will conduct measurements over product lifetime in multiple locations, understanding reproducibility critical. To determine the continuous-labeling, bottom-up...
Ammonium sulfate particles in air with average diameters ranging from 0.1 to 0.5‐μm have been generated by atomizing aqueous solutions of (NH 4 ) 2 SO various concentrations at ambient temperatures and pressures. The infrared spectra 4000 600 cm −1 the resulting aerosols investigated. This spectral region has allowed us study four infrared‐active vibrational modes this salt: ν 3 + ), (SO 2− ). frequencies these are similar published results obtained studies single crystal but displaced...
RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans effective at protecting rhesus macaques against lethal-dose aerosolized exposure. We previously used panel of RTA-specific monoclonal antibodies (MAbs) demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), elicits similar serum antibody profiles macaques. However, the MAb binding sites on have yet defined. In this study, we employed hydrogen...
Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, and many cases are important for protein function. The analysis of dynamics relies on biophysical techniques that can distinguish simultaneously existing populations molecules their rates interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding motions revealing unfolding wide timescale, ranging from seconds hours days. In this review we discuss HX...
Measurement of amide H/D exchange on the ms time scale can provide valuable information about dynamic behavior most flexible regions proteins. We describe here a simple mixing apparatus, assembled solely from off-the-shelf components, that be used for mass spectrometry to measure 50-5000 scale. Our apparatus utilizes flow-injection minimize sample consumption. Although mixer operates at low Reynolds numbers (less than 10(2)) where laminar flow is expected, kinetics were well-approximated...
ABSTRACT We previously produced a heavy-chain-only antibody (Ab) VH domain (V H H)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic toxin A subunit (RTA) binding B (RTB) (D. J. Vance, M. Tremblay, N. Mantis, C. B. Shoemaker, J Biol Chem 288:36538–36547, 2013, https://doi.org/10.1074/jbc.M113.519207 ). Initial subsequent screens by direct enzyme-linked immunosorbent assay (ELISA) yielded more than dozen unique RTA-...
Loss of deuterium label during the LC step in amide hydrogen/deuterium exchange mass spectrometry (H/D-MS) is minimized by maintaining an acidic mobile phase pH and low temperature (pH 2.5, 0 °C). Here we detail construction performance a low-cost, thermoelectrically refrigerated enclosure to house high-performance liquid chromatography (HPLC) components cool phases. Small volume heat exchangers rapidly decrease keep stable ±0.2 °C. Using superficially porous reversed-phase column, obtained...