McDonagh Brian, Martínez-Acedo Pablo, Vázquez Jesús, Padilla C Alicia, Sheehan David, Bárcena José Antonio
Department of Biochemistry and Molecular Biology, University of Córdoba and IMIBIC, 14071 Córdoba, Spain.
Int J Proteomics. 2012;2012:514847. doi: 10.1155/2012/514847. Epub 2012 Jul 15.
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 H(2)O(2) and also in vivo to the complex proteome of the Gram-negative bacterium Bacillus subtilis.
半胱氨酸是使用频率最低的氨基酸之一，但在蛋白质中保守存在时，它们通常在结构、功能或调节方面发挥关键作用。半胱氨酸的可逆修饰允许对蛋白质进行潜在的氧化还原调节。传统方法测量两个样品之间蛋白质的相对绝对量并不总是与蛋白质的活性成正比。我们建议将iTRAQ试剂与先前的硫醇选择方法相结合，以便在一次分析中相对定量样品内部和样品之间半胱氨酸的氧化还原状态。我们的方法能够鉴定蛋白质，识别蛋白质内对氧化还原敏感的半胱氨酸，并定量单个含半胱氨酸肽段的氧化还原状态。作为原理验证，我们将该技术应用于体外暴露于H₂O₂的酵母乙醇脱氢酶-1，以及体内革兰氏阴性细菌枯草芽孢杆菌的复杂蛋白质组。
