cysTMTRAQ—An Integrative Method for Unbiased Thiol-based Redox Proteomics

Abstract

Protein redox regulation plays important roles in many biological processes. Protein cysteine thiols are sensitive to redox changes and may function as redox switches, which turn signaling and metabolic pathways on or off to ensure speedy responses to environmental stimuli or stresses. Here we report a novel integrative proteomics method called cysTMTRAQ that combines two types of isobaric tags, cysteine tandem mass tags and isobaric tag for relative and absolute quantification, in one experiment. The method not only enables simultaneous analysis of cysteine redox changes and total protein level changes, but also allows the determination of bona fide redox modified cysteines in proteins through the correction of protein turnover. Changes in the redox states of protein cysteine thiols serve as regulatory switches in diverse biological processes (1). The redox cycle is regulated by well-known factors such as the ferredoxin-thioredoxin and glutathione-glutaredoxin systems, which reduce oxidized cysteines. Other oxidoreductases and oxidants such as reactive oxygen species act primarily to oxidize cysteine thiol groups (2, 3). In order to map and quantify cysteine redox modifications on the proteome scale, several approaches and methods have been developed, mostly using thiol-specific reagents and isotope tags. Twodimensional gel electrophoresis technology combined with fluorescent dye labeling (e.g. monobromobimane (4, 5) and cyanine dyes (6, 7)) and gel-free technology with isotope tagging (e.g. isotope-coded affinity tagging (6 -8), cysTMT 1 (9), and iTRAQ labeling of enriched cysteine-containing peptides (10 -14)) are often used to identify potential redox-sensitive cysteine residues and quantify redox changes.In addition to the well-known capabilities and limitations associated with two-dimensional gel electrophoresis-based and gel-free approaches (15), each method has its strengths and weaknesses in redox proteomics. For example, the twodimensional gel electrophoresis methods allow the inspection of spot patterns related to redox and protein-level changes. However, spot-volume-based quantification becomes problematic, as each spot often contains more than one protein species from complex samples. In addition, the limited number of fluorescent reagents compromises multiplexing capability, and the use of cyanine dyes does not allow mapping of the modified cysteines (6, 7). Other thiol labeling approaches such as the use of N-ethylmaleimide, biotin-N-[6-(Biotinamido)hexyl]-3-(2-pyridyldithio) propionamide (16), and isotopecoded affinity tags allow specific enrichment of cysteinecontaining peptides, mapping of cysteine modification sites, and duplex experiments in the case of isotope-coded affinity tags (6, 7). To enable multiplexing, 4-or 8-plex iTRAQ tags were recently used to label cysteine-containing peptides isolated from thiol-affinity chromatography (10,11,14,16). Another multiplexing technology, cysTMT, was developed to specifically label cysteines with free thiol groups of proteins from six different sampl...

Global Analysis of Myocardial Peptides Containing Cysteines With Irreversible Sulfinic and Sulfonic Acid Post-Translational Modifications

“…The cartridge was then washed with organic wash buffer (5 mM KH 2 PO 4 , pH 2.5, 25% MeCN) and this was collected separately (fraction "MeCN"). Retained peptides were eluted (each 1 ml) with varying concentrations of 100% KCl buffer (5 mM KH 2 PO 4 , pH 2.5, 25% MeCN, and 0.5 M KCl) diluted in organic wash buffer to 1,5,10,15,20,25,30,40,50,60,80, and 90% KCl. All fractions containing MeCN were dried by ϳ 1 ⁄2 -3 ⁄4 to reduce the MeCN concentration and then diluted with 0.1% TFA to Ͼ 1 ml before concentration and desalting by SPE as above.…”

“…spectral counting), MS precursor area, or for those compatible cell and tissue types, label-based quantitation that does not alter the charge or (49), which specifically target a small subset of sites for label-free quantitation, but requires prior knowledge of the modified sites. Recent work has tried to eliminate some of these problems for nonmodified/reversibly modified redox sites by employing cysTMTRAQ (50) to determine changes in redox-modified peptide abundance with respect to protein turnover. To our knowledge, this is also the only proteomics study to enrich Cys-SO 2 H/SO 3 H-containing peptides from tissue, which had not been subjected to exogenous oxidant addition.…”

Quantifying Reversible Oxidation of Protein Thiols in Photosynthetic Organisms

“…Although counterintuitive, protein thiols decreasing in level of oxidation upon oxidative stress is documented [39,43,44]. Our total coverage of 8592 cysteine-containing peptides compares favorably with other methods for quantification of reversible oxidation of protein thiols: 1098 cysteinyl peptides from Arabidopsis for OxiTRAQ [39], 912 cysteinyl peptides from E. coli for cysTMTRAQ [45], and 2067 cysteinyl peptides from Cyanobacteria for TPS6b coupled to iTRAQ quantitation [19]. Thus, these results indicate TPS6b coupled to label-free quantification is a robust and viable alternative for the analysis of reversible oxidation of protein thiols.…”

High-throughput screening of cellular redox sensors using modern redox proteomics approaches

“…Using the optimized ICAT-based method, Tpx1 and Pap1 were identified as redox-sensitive proteins involved in yeast fission [101]. CysTMTRAQ is another novel method combining TMT and isobaric tags for quantification, thus allowing the simultaneous analysis of the redox states of thiols and the global protein level [104,108]. In order to both identify variations of protein expression and analyze the percentage of redox modification, Jackson's group have reported a label-free quantitative proteomic approach that includes a differential cysteine labeling step [109].…”