2.6. Redox proteomic analysis

Redox proteomics was performed using redox isotope coded affinity tag (ICAT)-based mass spectrometry [20, 52, 53]. Briefly, freshly harvested lung tissue from mice exposed to Cd or vehicle control (n = 3/group) were homogenized in ice-cold 10% TCA. Protein precipitate was washed with ice-cold acetone, resuspended in denaturing buffer (50 mM Tris, 0.1% SDS, pH 8.5). Reduced cysteine (Cys) of proteins were labeled with the biotin-conjugated thiol reagent (Heavy isotopic [H-ICAT]). Proteins were then reprecipitated by ice-cold 10% TCA for 30 min on ice, pelleted, washed with acetone, and resuspended in denaturing buffer. Minimally oxidized (sulfenic acid) and disulfides in the proteins were then reduced by TCEP [tris-(2-carboxyethyl phosphine)] and labeled with another biotin-conjugated thiol reagent (Light isotopic [L-ICAT]). Samples including both H and L-ICAT-labeled Cys residues in proteins were digested with trypsin overnight, fractionated by cationic exchange followed by avidin purification, and analyzed by mass spectrometer as described below. ICAT-labeled Cys containing peptides (peptidyl Cys) were identified with an H to L ratio as a measure of the redox (reduced/oxidized) state of the protein, expressed as “% oxidation of protein”.

ICAT-labeled Cys peptides were analyzed by reverse-phase LC-MS/MS [18, 20, 54] using a LTQ-Orbitrap ion trap mass spectrometer (Thermo) (300–1600 m/z). The acquired MS/MS spectra were searched against a concatenated target-decoy mouse reference database of the National Center for Biotechnology Information using the SEQUEST Sorcerer algorithm [55]. The peptides were classified by charge state and tryptic state (fully and partial) and first filtered by mass accuracy (10 ppm for high-resolution MS) and then dynamically by increasing XCorr and Cn values to reduce protein false discovery rate (FDR) to less than 1%, according to the target-decoy strategy [56].