Abstract
Cysteines are one of the most rarely used amino acids, but when conserved in proteins they often play critical roles in structure, function, or regulation. Reversible cysteine modifications allow for potential redox regulation of proteins. Traditional measurement of the relative absolute quantity of a protein between two samples is not always necessarily proportional to the activity of the protein. We propose application of iTRAQ reagents in combination with a previous thiol selection method to relatively quantify the redox state of cysteines both within and between samples in a single analysis. Our method allows for the identification of the proteins, identification of redox-sensitive cysteines within proteins, and quantification of the redox status of individual cysteine-containing peptides. As a proof of principle, we applied this technique to yeast alcohol dehydrogenase-1 exposed in vitro to H2O2 and also in vivo to the complex proteome of the Gram-negative bacterium Bacillus subtilis.
Highlights
The dynamic nature of the proteome ensures that the cell is able to respond to perturbation of environmental, genetic, biochemical, and pathological conditions
Inspection of the results indicates that under control conditions, approximately half of these thiols were reversibly oxidized (47%) and half were in a reduced state (53%)
The average pKa value of cysteines has been calculated as 6.8 ± 2.7, indicating that at physiological pH, they may exist in both charged thiolate form and uncharged form depending on a number of factors [26, 27]
Summary
The dynamic nature of the proteome ensures that the cell is able to respond to perturbation of environmental, genetic, biochemical, and pathological conditions. Improvements in mass spectrometry has led to the development of a number of techniques to quantify the relative protein abundance within a given sample. These include isotope-coded affinity tags (ICATs) [1], stable isotope labeling of amino acids in cell culture (SILAC) [2], and isobaric tags for relative and absolute quantification (iTRAQ) [3]. Measuring the relative quantity of a protein between two samples does not tell us anything about the activity of the protein itself. This is especially important in reference to redox proteins that contain thiol switches susceptible to activation or inactivation
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