Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

10.3791/61983 ◽  
2021 ◽  
Author(s):  
Zachary J. Kirsch ◽  
Blaise G. Arden ◽  
Richard W. Vachet ◽  
Patanachai Limpikirati
Keyword(s):  
Mass Spectrometry ◽  
Higher Order ◽  
Order Structure ◽  
Covalent Labeling

Covalent Labeling Denaturation Mass Spectrometry for Sensitive Localized Higher Order Structure Comparisons

2016 ◽  
Vol 88 (4) ◽  
pp. 2478-2488 ◽  
Author(s):  
James A. Madsen ◽  
Yan Yin ◽  
Jing Qiao ◽  
Vanessa Gill ◽  
Kutralanathan Renganathan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Higher Order ◽  
Order Structure ◽  
Covalent Labeling

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.

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