Identification by MS/MS of disulfides produced by a functional redox transition.
Among posttranslational modifications of proteins entailed with signal transduction, the redox transition is today brought to the focus as a major biochemical event accounting for the signaling functions of reactive oxygen species. Thermodynamic and kinetic criteria highlight hydroperoxides and protein disulfides as signaling and transducer elements, respectively, and growing biochemical evidence supports this notion. The protein Cys residue involved in this function must react fast and specifically with the oxidant and then with a second accessible Cys yielding the disulfide. These kinetic and structural constraints are shared with peroxidases and peroxiredoxins, which are competitors for the signaling hydroperoxide. In this chapter, a procedure based on MS/MS analysis for inter- and intrachain disulfide assignment in proteins undergoing redox-switch is presented. While the sensitivity of the modern MS/MS instruments permits the sequencing of double peptides linked by a disulfide bond, the major pitfall of the proteomic procedure is the thiol-disulfide scrambling taking place at the alkaline pH needed for the proteolytic reaction of trypsin. Instead, the use of pepsin at acidic pH prevents the disulfide scrambling, but the specificity of the proteolytic reaction is low and thus the complexity of fragmentation increases. We succeeded to limit this problem by heuristically assuming a conserved pepsin cleavage pattern of the protein both in the oxidized and the reduced form. Asymmetric cleavage of the disulfide by collisional fragmentation further corroborated the identification. In conclusion, the use of pepsin, integrated by a minimal computation, appears suitable for positively assigning inter- and intrachain disulfides generated by a functional redox-switch.
Institute for Biomedical Technologies, National Research Council, Viale Fratelli Cervi, Segrate-Milano, Italy.
Pierluigi
P
Mauri
Stefano
S
Toppo
Antonella
A
De Palma
Louise
L
Benazzi
Matilde
M
Maiorino
Fulvio
F
Ursini
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D003627:Data Interpretation, Statistical; D004220:Disulfides / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004220:Disulfides