Concentration of Hydroxyl Radicals during Electrical Discharge for Radical Probe Protein Footprinting Mass Spectrometry

2019 ◽  
Author(s):  
Kevin.M. Downard
Keyword(s):  
Mass Spectrometry ◽  
Hydroxyl Radicals ◽  
Electrical Discharge ◽  
Protein Footprinting

Protein Footprinting with Radical Probe Mass Spectrometry- Two Decades of Achievement

2019 ◽  
Vol 26 (1) ◽  
pp. 4-15 ◽  
Author(s):  
Simin D. Maleknia ◽  
Kevin M. Downard
Keyword(s):  
Mass Spectrometry ◽  
Hydroxyl Radicals ◽  
Protein Structures ◽  
Protein Docking ◽  
Photochemical Oxidation ◽  
Annual Conference ◽  
Protein Footprinting ◽  
X Ray ◽  
American Society ◽  
The Impact

Background: Radical Probe Mass Spectrometry (RP-MS) describes a pioneering methodology in structural biology that enables the study of protein structures, their interactions, and dynamics on fast timescales (down to sub-milliseconds). Hydroxyl radicals (•OH) generated directly from water within aqueous solutions induce the oxidation of reactive, solvent accessible amino acid side chains that are then analyzed by mass spectrometry. Introduced in 1998 at the American Society for Mass Spectrometry annual conference, RP-MS was first published on in 1999. Objective: This review article describes developments and applications of the RP-MS methodology over the past two decades. Methods: The RP-MS method has been variously referred to as synchrotron X-ray radiolysis footprinting, Hydroxyl Radical Protein Footprinting (HRPF), X-ray Footprinting with Mass Spectrometry (XF-MS), Fast Photochemical Oxidation of Proteins (FPOP), oxidative labelling, covalent oxidative labelling, and even the Stability of Proteins from Rates of Oxidation (SPROX). Results: The article describes the utility of hydroxyl radicals as a protein structural probe, the advantages of RP-MS in comparison to other MS-based approaches, its proof of concept using ion mobility mass spectrometry, its application to protein structure, folding, complex and aggregation studies, its extension to study the onset of protein damage, its implementation using a high throughput sample loading approach, and the development of protein docking algorithms to aid with data analysis and visualization. Conclusion: RP-MS represents a powerful new structural approach that can aid in our understanding of the structure and functions of proteins, and the impact of sustained oxidation on proteins in disease pathogenesis.

Current Trends in Biotherapeutic Higher Order Structure Characterization by Irreversible Covalent Footprinting Mass Spectrometry

2019 ◽  
Vol 26 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Natalie K. Garcia ◽  
Galahad Deperalta ◽  
Aaron T. Wecksler
Keyword(s):  
Mass Spectrometry ◽  
Protein Structure ◽  
Protein Interactions ◽  
Quaternary Structure ◽  
Solvent Accessibility ◽  
Higher Order ◽  
Structure Characterization ◽  
Order Structure ◽  
Protein Footprinting ◽  
Wide Range

Background: Biotherapeutics, particularly monoclonal antibodies (mAbs), are a maturing class of drugs capable of treating a wide range of diseases. Therapeutic function and solutionstability are linked to the proper three-dimensional organization of the primary sequence into Higher Order Structure (HOS) as well as the timescales of protein motions (dynamics). Methods that directly monitor protein HOS and dynamics are important for mapping therapeutically relevant protein-protein interactions and assessing properly folded structures. Irreversible covalent protein footprinting Mass Spectrometry (MS) tools, such as site-specific amino acid labeling and hydroxyl radical footprinting are analytical techniques capable of monitoring the side chain solvent accessibility influenced by tertiary and quaternary structure. Here we discuss the methodology, examples of biotherapeutic applications, and the future directions of irreversible covalent protein footprinting MS in biotherapeutic research and development. Conclusion: Bottom-up mass spectrometry using irreversible labeling techniques provide valuable information for characterizing solution-phase protein structure. Examples range from epitope mapping and protein-ligand interactions, to probing challenging structures of membrane proteins. By paring these techniques with hydrogen-deuterium exchange, spectroscopic analysis, or static-phase structural data such as crystallography or electron microscopy, a comprehensive understanding of protein structure can be obtained.

scholarly journals Probing Conformational Changes of Human DNA Polymerase λ using Mass Spectrometry-Based Protein Footprinting

2009 ◽  
Vol 390 (3) ◽  
pp. 368-379 ◽  
Author(s):  
Jason D. Fowler ◽  
Jessica A. Brown ◽  
Mamuka Kvaratskhelia ◽  
Zucai Suo
Keyword(s):  
Mass Spectrometry ◽  
Dna Polymerase ◽  
Conformational Changes ◽  
Protein Footprinting ◽  
Human Dna ◽  
Dna Polymerase Λ

scholarly journals Mass Spectrometry-Based Protein Footprinting Characterizes the Structures of Oligomeric Apolipoprotein E2, E3, and E4

Biochemistry ◽  
2011 ◽  
Vol 50 (38) ◽  
pp. 8117-8126 ◽  
Author(s):  
Brian Gau ◽  
Kanchan Garai ◽  
Carl Frieden ◽  
Michael L. Gross
Keyword(s):  
Mass Spectrometry ◽  
Protein Footprinting

Hybrid Gas/Liquid Electrical Discharge Reactors with Zeolites for Colored Wastewater Degradation

2005 ◽  
Vol 8 (2) ◽  
Author(s):  
Hrvoje Kušić ◽  
Natalija Koprivanac ◽  
Igor Peternel ◽  
Bruce R. Locke
Keyword(s):  
Hydrogen Peroxide ◽  
Liquid Phase ◽  
Gas Phase ◽  
Hydroxyl Radicals ◽  
Electrical Discharge ◽  
Organic Dye ◽  
Electrical Discharges ◽  
Colored Wastewater ◽  
Ozone Levels ◽  
Parallel Reactor

AbstractHybrid gas/liquid electrical discharge reactors have been used to degrade an organic dye in the presence and absence of zeolites. Simultaneous gas and liquid phase electrical discharges in the hybrid parallel and hybrid series reactors have been shown in previous work to lead to the formation of hydrogen peroxide and hydroxyl radicals in the liquid phase and ozone in the gas phase. These reactors differ in their electrode configuration, and in previous work it was shown that the ozone levels in the parallel reactor are seven times higher than in the series reactor (3000 ppm and 450 ppm, respectively), while both reactors produce the same levels of hydrogen peroxide (4.9 × 10

scholarly journals Validated Determination of NRG1 Ig-like Domain Structure by Mass Spectrometry Coupled with Computational Modeling

2021 ◽  
Author(s):  
Niloofar Abolhasani Khaje ◽  
Alexander Eletsky ◽  
Sarah E. Biehn ◽  
Charles K. Mobley ◽  
Monique J. Rogals ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Structure ◽  
Computational Modeling ◽  
Solvent Exposure ◽  
Uncharacterized Protein ◽  
Single Experiment ◽  
Protein Footprinting ◽  
Hydroxyl Radical Protein Footprinting ◽  
Unknown Structure

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally-informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure leaving questions of unintentional bias and applicability to unknown structures unresolved. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 Å. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure.

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