Abstract
Functional disulfide bonds mediate a change in protein function in which they reside when cleaved or formed. To elucidate how a functional disulfide bond controls protein activity, it is critical that the redox state of the bond in the population of protein molecules is known. Measurement of changes in disulfide bond redox state relies on thiol probes and immunoblotting. Such technique only offers a qualitative indication of a change in redox state but not the identity of cysteines involved. A differential cysteine alkylation and mass spectrometry technique is described here that affords precise quantification of protein disulfide bond redox state. The utility of the technique is demonstrated by quantifying the redox state of 24 of the 28 disulfide bonds in human β3 integrin from purified platelets.
Keywords: Disulfide bonds, Redox, Alkylation, Proteomics, Integrin
Background
Disulfide bonds are covalent linkages between two cysteine residues in a protein. There are three types of disulfide bonds found in proteins: structural, catalytic and functional. Structural disulfide bonds stabilize protein conformation and are the most common type disulfide bonds. Catalytic disulfide bonds are found in redox enzymes such as thioredoxin and protein disulfide isomerases that reduce and oxidize disulfide bonds in substrate proteins. Functional disulfide bonds mediate a change of protein function such as activity, ligand affinity, localization or multimerization when cleaved or formed. Our understanding of how functional disulfides control protein function is limited by the molecular techniques available to detect changes in cysteines and disulfide bonds. Measurement of changes in disulfide redox state has mainly relied on the use of thiol probes such as biotin-conjugated maleimide (3-(N-maleimidylpropionyl)biocytin), followed by detection using streptavidin conjugated-peroxidase (Manickam et al., 2008). This provides a qualitative indication of a change in redox state and does not identify which cysteines are involved. The development of isotopic cysteine alkylators (unlabeled and 13Carbon labeled) has allowed differential cysteine alkylation, and mass spectrometry allows for an estimation of the fraction of a protein disulfide bond that is reduced in the protein preparation (Chambers et al., 2010). In the method described herein, reduced disulfide bond cysteines are alkylated with 2-iodo-N-phenylacetamide (12C-IPA) and the oxidized disulfide bond cysteines with a stable carbon-13 isotope of IPA (13C-IPA) following reduction with dithiothreitol. The ratio of alkylation of peptides containing the disulfide bond cysteines with 12C-IPA compared to 13C-IPA represents the fraction of the disulfide in the population that is in the reduced state. The advantage of this pair of cysteine alkylators is that they have the same chemical reactivity and the same structure, which enhances the reliability of alkylation, resolution of the alkylated peptides by liquid chromatography and their detection by mass spectrometry (Pasquarello et al., 2004). A mass difference of 6 Da is the only change in a cysteine labeled with 12C-IPA or 13C-IPA. To exemplify this technique, the redox state of disulfide bonds in β3 integrin from platelet is calculated. Based on the published crystal structure (PDB: 3FCS), there are 56 cysteines that form 28 disulfide bonds in β3 integrin. Using this protocol, sixty-eight IPA-labeled peptides encompassing 34 of the 56 cysteines in the β3 integrin subunit were resolved, which represents 24 of the 28 β3 integrin disulfide bonds. Among the 24 disulfide bonds measured, one disulfide bond, Cys177-184, is subjected to cleavage by disulfide-cleaving enzyme ERp5, thereby regulating ligand affinity of β3 integrin on platelet surface (Passam et al., 2018).
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Acknowledgments
This work was supported by the Helen and Robert Ellis Fellowship and Tony Basten Postdoctoral Fellowship from the Sydney Medical School Foundation. This work is adapted from previous work (Passam et al., 2018).
Competing interests
The author declares no conflict of interest.
References
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