Large-Scale Capture of Peptides Containing Reversibly Oxidized Cysteines by Thiol-Disulfide Exchange Applied to the Myocardial Redox Proteome

Abstract

Redox regulation is emerging as an important post-translational modification in cell signaling and pathogenesis. Cysteine (Cys) is the most redox active of the commonly coded amino acids and is thus an important target for redox-based modifications. Reactions that oxidize the Cys sulfur atom to low oxidation states (e.g., disulfide) are reversible, while further reactions to higher oxidation states (e.g., sulfonic acid) may be irreversible under biological conditions. Reversible modifications are particularly interesting as they mediate redox signaling and regulation of proteins under physiological conditions and during adaptation to oxidant stress. An enrichment method that relied on rapid and specific alkylation of free Cys, followed by thiol-based reduction and resin capture by thiol-disulfide exchange chemistry was applied to isolate reversibly modified Cys-containing peptides. Chromatographic conditions were optimized to provide increased specificity by removal of noncovalent interactions. The technique was highly efficient, based on near equimolar reactions with the resin, reproducible and linear for peptide elution, as quantified by label-free mass spectrometry. The method was applied to a complex protein lysate generated from rat myocardial tissue and 6559 unique Cys-containing peptides from 2694 proteins were identified. Comparison with the rat database and previous studies showed effective enrichment of proteins modified by S-nitrosylation, disulfide formation, and Cys-sulfenic acid. Analysis of amino acid sequence features indicated a preference for acidic residues and increased hydrophilicity in the regions immediately up- or downstream of the reactive Cys. This technique is ideally suited for the enrichment and profiling of reversible Cys modifications on a proteome-wide scale.

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

“…Lewis rats (n ϭ three per group) were euthanized with pentobarbital (200 mg kg Ϫ1 ), the heart rapidly ex-cised and subjected to Langendorff perfusion for 20 min ("baseline" perfusion) with nonrecirculating Krebs buffer as previously described (39), followed by either; 1) 30 min of perfusion (nonischemic time control, NITC), or 2) 15 min of global no-flow ischemia and 15 min of reperfusion (15I/15R). Hemodynamic data based on the rate pressure product (RPP; a function of the heart rate in bpm ϫ left ventricular developed pressure [LV dev ]) expressed as a percentage of baseline showed NITC hearts were unaffected by control perfusion (RPP 105% Ϯ 1% of baseline), whereas those subjected to 15I/15R recovered to 66% Ϯ 5% of baseline, consistent with previous studies (7,39). Hearts were snap-frozen in liquid nitrogen and stored at Ϫ80°C.…”

Quantifying Reversible Oxidation of Protein Thiols in Photosynthetic Organisms

“…In Arabidopsis, we identified 91000 unique cysteinyl peptides at 84.8%-90.2% enrichment efficiency across five enrichment replicates (Supplemental Table 4). In comparison, TPS6b coupled to LFQ identified 1988 unique peptides in rat myocardial tissue at 94% enrichment [16], whereas TPS6b coupled to iTRAQ identified 670 nitrosylated peptides in cyanobacteria at 995% enrichment [18].…”

“…TPS6b yields excellent coverage (91000 cysteinyl peptides) when coupled to label-free or tagged quantitation in cyanobacteria and rat myocardium [16,18]. Here, we evaluated coverage, efficiency, and precision of TPS6b in photosynthetic cells/tissues coupled to label-free MS by splitting protein lysates of C. reinhardtii or Arabidopsis into the three or five 0.5 mg aliquots and performing separate enrichments (Figure 2).…”

“…Although this approach has been used to profile sites of nitrosylation in Arabidopsis [11], glutathiolylation in cyanobacteria [12], and oxidation in tomato [13], optimization of enrichment efficiency remains a key concern as background noise is high in biotin-switch assays [14] and an optimized protocol for cysTMT using a competitive elution buffer only contained 21% cysteinyl peptides [15]. Alternatively, instead of tagging oxidized thiols after reduction, affinity purification can proceed directly for the now free thiols using a disulfide exchange resin, Thiopropyl Sepharose 6B (TPS6b) [16]. Initially used to increase depth of proteome coverage for discovery experiments [17], TPS6b has recently been used for redox proteomics in cyanobacteria [18,19] and rat myocardium [16] at enrichment efficiencies 995%.…”

“…Alternatively, instead of tagging oxidized thiols after reduction, affinity purification can proceed directly for the now free thiols using a disulfide exchange resin, Thiopropyl Sepharose 6B (TPS6b) [16]. Initially used to increase depth of proteome coverage for discovery experiments [17], TPS6b has recently been used for redox proteomics in cyanobacteria [18,19] and rat myocardium [16] at enrichment efficiencies 995%.…”