Isotope-coded Affinity Tag Approach to Identify and Quantify Oxidant-sensitive Protein Thiols

Abstract

An approach is described for identifying and quantifying oxidant-sensitive protein thiols using a cysteine-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, Foster City, CA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT reagent, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. To validate our approach, creatine kinase with four cysteine residues, one of which is oxidant-sensitive, was chosen as an experimental model. ICAT-labeled peptides derived from creatine kinase were used to evaluate the relative abundance of the free thiols in samples subjected (or not) to treatment with hydrogen peroxide. As predicted, hydrogen peroxide decreased the relative abundance of the unmodified oxidant-sensitive thiol residue of cysteine-283 in creatine kinase, providing proof of principle that an ICAT-based quantitative mass spectrometry approach can be used to identify and quantify oxidation of cysteine thiols. This approach opens an avenue for proteomics studies of the redox state of protein thiols. Regulation of cellular homeostasis through post-translational modification of proteins is one of the major responses to oxidative and nitrosative stress (1). Proteins containing cysteine thiol groups are particularly susceptible to oxidation by free radicals, electrophilic molecules, and nitric oxide donors (2, 3). One or more reduced thiol groups are essential for the function of many proteins. Oxidation of these critical thiol groups can increase or decrease the activity of these proteins and represents not only a major mechanism of normal cell signaling via S-nitrosation (4) or S-glutathiolation (5), but also a mechanism by which disease and aging interferes with protein function by irreversible thiol oxidation (6). Thus, it is essential to identify the proteins containing Cys residues and their relative sensitivity to oxidation. As a step in the development of a proteomic approach to identify post-translational modification of Cys residues in proteins involved in redox signaling or those affected by disease, this report describes a method to identify and quantify oxidant-sensitive protein thiols by mass spectrometric peptide fingerprinting.The procedure is based on the fact that oxidized Cys residues are not susceptible to modification by iodoacetamide (IAM) 1 analogues (7). Isotope-coded affinity tag (ICAT) reagents that are IAM analogues have been used extensively in quantitative proteomics to evaluate the abundance of expressed proteins (8). The ICAT approach is based on affinity tag targeting of free cysteines in proteins that are labeled after the proteins are isolated under strong reducing conditions. This study was designed to determine the feasibility of using the acid-cleavable IAM-based ICAT reagent (catalogue no. 4337335; Applied Biosystems, Foster City, CA) to quantitate the extent of thiol oxidation under nonreducing conditions. The principle of the ICAT app...

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo

“…MS/MS analysis of the peptide is used to identify the respective protein and, at the same time, the oxidation sensitive cysteine(s). Although Sethuraman and coworkers have previously used ICAT technology to determine relative changes in the availability of free protein thiols in two separate samples (13,15,16), the OxICAT method provides the absolute ratio of reduced to oxidized protein within a single sample. This makes OxICAT also ideally suited for analyzing changes in the redox status of proteins in cells exposed to stress conditions, because neither changes in protein expression nor protein stability influence the OxICAT result.…”

“…The recent interest in redox-regulated proteins fueled by the increasing recognition of their important physiological roles however, led to the development of several global thiol trapping techniques (10)(11)(12)(13). These methods, albeit capable of detecting proteins with redox-sensitive cysteines in lysates and intact cells, often lack the ability to directly identify the proteins or cysteines involved, and more importantly, to quantify the extent of oxidative thiol modifications.…”

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…When isotope-coded-affinitytags (ICAT) reagents are used in this way, relative quantification between two differentially oxidised samples can be obtained [113,114]. These techniques are useful to identify oxidation-sensitive thiols, but fail to identify the type of oxidation.…”

Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

“…The degree of oxidation is quantified from the decrease in labeling calculated from the ratio of ICAT labeling of control and oxidant-modified samples (Figure 2). The modified cysteine residue is identified from the liquid chromatography (LC) mass spectrometry (MS and MS/ MS) analysis of the peptides obtained after proteolysis of the protein(s) [22,23]. For the present experiments we used ONOO − and GSSG which regulate the activity of p21ras by introducing various oxidative modifications [17,19].…”