A5062508980- Mass Spectrometry Techniques and Applications
- Protein purification and stability
- Analytical Chemistry and Chromatography
- Advanced Proteomics Techniques and Applications
- Monoclonal and Polyclonal Antibodies Research
- Cholesterol and Lipid Metabolism
- Lipid Membrane Structure and Behavior
- Microfluidic and Capillary Electrophoresis Applications
- Antimicrobial Peptides and Activities
- Histone Deacetylase Inhibitors Research
- Pharmacogenetics and Drug Metabolism
- Chemical Synthesis and Analysis
- Viral Infectious Diseases and Gene Expression in Insects
- Hormonal Regulation and Hypertension
- Glycosylation and Glycoproteins Research
- Epigenetics and DNA Methylation
- Cannabis and Cannabinoid Research
- Genetics and Neurodevelopmental Disorders
- World Wars: History, Literature, and Impact
- Photography and Visual Culture
- Blood transfusion and management
- Inflammatory mediators and NSAID effects
- Ion Transport and Channel Regulation
- Mosquito-borne diseases and control
- Metabolomics and Mass Spectrometry Studies
National Institute of Standards and Technology
2015-2024
National Institute of Standards
2024
Material Measurement Laboratory
2015-2024
Institute for Bioscience and Biotechnology Research
2014-2024
University of Maryland, College Park
2012-2024
Research Institute for Bioscience and Biotechnology
2024
Advanced Bioscience Laboratories (United States)
2012-2021
University of Maryland, Baltimore
2015-2021
Histone deacetylase (HDAC) inhibition has promise as a therapy for Alzheimer's disease (AD) and other neurodegenerative diseases. Currently, therapeutic HDAC inhibitors target many isoforms, particularly detrimental approach when isoforms are known to have different specialized functions. We developed multiple reaction monitoring (MRM) mass spectrometry assay using stable isotope-labeled QconCATs internal standards quantify isoforms. further determined quantitative pattern of specific HDACs...
Alzheimer's disease (AD) is the sixth leading cause of death and most costly in US. Despite enormous impact AD, there are no treatments that delay onset or stop progression currently on market. This partly due to complexity largely unknown pathogenesis sporadic which accounts for vast majority cases. Epigenetics has been implicated as a critical component AD pathology potential "hot spot" treatments. Histone post-translational modifications (PTMs) key element epigenetic regulation gene...
Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls elimination from the brain. This also potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified efavirenz, anti-HIV medication. Previously, we suggested pharmaceuticals activate CYP46A1 allosterically through binding to site on cytosolic protein surface, which different active facing membrane. Here identified this allosteric...
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...
Quantification by targeted proteomics has largely depended on mass spectrometry and isotope-labeled internal standards. In addition to traditionally used recombinant proteins or synthetic peptides, concatenated peptides (QconCATs) were introduced as a conceptually new source of standard. the present study, we focused assessing length natural flanking sequences, which surround each peptide included in QconCAT provide for identical rates analyte standard digestion trypsin. We have expressed,...
Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereospecific hydroxylation of different sterols. In humans, complete CYP27A1 deficiency leads to cerebrotendinous xanthomatosis nodule formation in tendons brain (preferentially the cerebellum) rich cholesterol cholestanol, 5α-saturated analog cholesterol. Cyp27a1-/- mice, xanthomas are not formed, despite significant cholestanol increase cerebellum. The mechanism behind...
Cytochrome P450 46A1 (CYP46A1) converts cholesterol to 24-hydroxycholesterol and thereby controls the major pathways of removal from brain. Cyp46a1-/- mice have a reduction in rate biosynthesis brain significant impairments memory learning. To gain insights into mechanisms underlying phenotype, we used quantified their sterol levels expression genes pertinent homeostasis. We also compared wild type brains for protein phosphorylation ubiquitination. The data obtained enable following...
In the search for novel broad-spectrum therapeutics to fight chronic infections, inflammation, and cancer, host defense peptides (HDPs) have garnered increasing interest. Characterizing their biologically-active conformations minimum motifs function represents a requisite step developing them into efficacious safe therapeutics. Here, we demonstrate that metallating HDPs with Cu2+ is an effective chemical strategy improve cytotoxicity on cancer cells. Mechanistically, find prepared as...
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 promote greater reproducibility. Standard HDX-MS analysis systems that digest proteins and separate peptides at pH 2.7 0 °C can lose >30% the deuterium marker within 15 min sample injection. This report describes architecture performance a dual-enzyme, instrument conducts liquid...
Developing new antimicrobials as alternatives to conventional antibiotics has become an urgent race eradicate drug-resistant bacteria and save human lives. Conventionally, antimicrobial molecules are studied independently even though they can be cosecreted in vivo. In this research, we investigate two classes of naturally derived antimicrobials: sophorolipid (SL) esters modified yeast-derived glycolipid biosurfactants that feature high biocompatibility low production cost; piscidins, which...
The absolute quantitation of amine metabolites from mammalian cell samples was achieved by combining standards, isobaric tags, and capillary liquid chromatography (LC) tandem mass spectrometry (MS/MS). Our approach allowed 32 specific amines to be analyzed within a single chromatographic run, with the generation calibration curve each analyte taking less than 900 ms. Using this strategy, we determined response human aortic endothelial cells (HAECs) glucose challenge. observed changes...
The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, most common tick-borne disease in United States. low abundance bacterial proteins human serum during infection imposes a challenge for early proteomic detection disease. To address this challenge, we propose to detect membrane released from bacteria due disruption their plasma triggered by innate immune system. These can be separated bulk high-speed centrifugation causing substantial sample enrichment prior targeted...
The increasing interest in extracellular vesicles (EVs) research is fueled by reports indicating their unique role intercellular communication and potential connection to the development of common human diseases. assumes protein nucleic acid cargo. Unfortunately, accurate analysis EVs cargo faces a challenge isolation. Generally used isolation techniques do not separate different subtypes even more, poorly from non-EVs contaminants. Further protocols urgently needs quantitative method purity...
Alzheimer's disease has been shown to have a global reduction in gene expression, called an epigenetic blockade, which may be regulated by histone post-translational modifications. Histone H3 highly phosphorylation. We, therefore, chose for investigation of phosphorylation the core sites serine-57 (S57) and threonine-58 (T58). Hemispheres brains from mouse model rapid amyloid deposition (5XFAD) were used measurement S57 T58 Multiple reaction monitoring (MRM) was measure level...
Lipid nanoparticles are a generic type of nanomaterial with broad applicability in medicine as drug delivery vehicles. Liposomes subtype lipid and, therapeutic platform, can be loaded genetic material or pharmaceutical agents for use treatments. An open question these types is what factor(s) affect the long-term stability particles. The particle great interest to understand and predict effective shelf-life storage requirements. In this report, we detail one-year study liposome function...
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 handle full-length membrane in lipid bilayers. Additionally, lipids used mimic environment and solubilize can impair chromatography performance cause ion suppression spectrometer. The workflow discussed herein advances HDX-MS capabilities other MS...
Chromatographic separations at subzero temperature significantly improve the precision of back-exchange-corrected hydrogen–deuterium exchange mass spectrometry (HDX-MS) determinations. Our previously reported dual-enzyme HDX-MS analysis instrument used reversed phase liquid chromatography (RPLC) −30 °C, but high backpressures limited flow rates and required materials equipment rated for very pressures. Here, we report design performance a comprising RPLC trap column hydrophilic interaction...
Abstract Inhibition of human Monoacylglycerol Lipase (hMGL) offers a novel approach for treating neurological diseases. The design inhibitors, targeting active-inactive conformational transitions the enzyme, can be aided by understanding interplay between structure and dynamics. Here, we report effects mutations within catalytic triad on structure, gating dynamics hMGL combining kinetics, NMR, HDX-MS data with metadynamics simulations. We found that point alter delicate equilibria active...
Previous reports present different models for the stabilization of Fc-FcγRI immune complex. Although accord exists on importance L235 in IgG1 and some hydrophobic contacts complex stabilization, discord regarding existence stabilizing glycoprotein between glycans a conserved FG-loop (171MGKHRY176) FcγRIa. Complexes formed from FcγRIa receptor IgG1s containing biantennary with N-acetylglucosamine, galactose, α2,6-N-acetylneuraminic terminations were measured by hydrogen-deuterium exchange...
Using mAbs as therapeutic molecules is complicated by the propensity of to aggregate at elevated concentrations, which can lead a variety adverse events in treatment. Here, we describe proof-of-concept for new methodology detect and quantify mAb aggregation. Assay development included using an aggregated bait screening phage display peptide library identifying those peptides with random sequence recognize aggregates. Once identified, selected be used developing quantitative methods assess...
PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn2+ or Mg2+ ions for activity. Whereas the crystal structure human PP2Cα displayed two tightly bound active site, recent investigations have characterized binding a third, catalytically essential metal ion. The third to was reported affinity be entropically driven, suggesting it may structurally important. Here, we report use hydrogen/deuterium exchange-mass spectrometry...
Sample carryover is a common problem in hydrogen-deuterium exchange mass spectrometry, particularly because immobilized protease columns cannot withstand the high organic solvent concentrations typically used liquid chromatography spectrometry (LC-MS) for cleaning. Conventional cleaning methods using injections of guanidine HCl still suffer from and may require four blanks after each sample run to fully remove carryover. We have implemented an additional LC pump deliver customized wash...