Functional Disulphide Bonds

This book describes the current techniques used to study functional disulphides and to exploit them for therapeutic outcomes. Beginning with how disulphide bonds are classified and how to study their evolution, the volume then continues with protein chemical, molecular dynamics, and force spectroscopy techniques used to identify functional disulphides and/or study their behavior, then focuses on techniques employed in the study of the factors that cleave and/or form disulphide bonds. Methods to study how particular functional disulphides are assayed in mouse and human tissues are likewise examined, amongst many other subjects. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, FunctionalDisulphide Bonds: Methods and Protocols serves as a vital guide to this evolving area of study.

Includes cutting-edge techniques Provides step-by-step detail essential for reproducible results Contains key implementation advice from the experts

Inhalt

1. Classification of protein disulfide bonds.- 2. Assessing the evolutionary conservation of protein disulphide bonds.- 3. A proteomics workflow for the Identification of labile disulphide bonds at the cell surface.- 4. Quantification of the redox state of protein disulfide bonds.- 5. Quantification of the redox state of protein disulfide bonds.- 6. Studying functional disulfide bonds by computer simulations.- 7. Dynamic force spectroscopy analysis on the redox states of protein disulfide bonds.- 8. Assays of thiol isomerase enzymatic activity.- 9. Functional assays of thiol isomerase ERp5.- 10. IDENTIFICATION OF PDI SUBSTRATES BY MECHANISM-BASED KINETIC TRAPPING.- 11. Site-specific proteomic mapping of modified cysteine residues.- 12. Quantitation of glutathione, glutathione disulfide and protein-glutathione mixed disulfides by high performance liquid chromatography-tandem mass spectrometry.- 13. Interrogation of Functional Mitochondrial Cysteine Residues by Quantitative Mass Spectrometry.- 14. Oxidative Protein Folding Using trans-3,4-Dihydroxyselenolane Oxide.- 15. Methodology for identification of the cysteine-reactive covalent inhibitors.- 16. In Vivo Measurement of Redox-Regulated TG2 Activity.- 17. Quantitation of total and free thiol 2-glycoprotein I levels for diagnostic and prognostic purposes in the antiphospholipid syndrome.- 18. Quantitation of total and free thiol angiotensinogen as a prognostic marker for pre-eclampsia.- 19. Preparation of a dithiol-reactive probe for PET imaging of cell death.- 20. Flow Cytometry Assessment of Procoagulant Platelets Using A Dithiol Reactive Probe.