Targeted Quantitation of Site-Specific Cysteine Oxidation in Endogenous Proteins Using a Differential Alkylation and Multiple Reaction Monitoring Mass Spectrometry Approach

Abstract

Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteine-specific redox-sensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the site-specific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase-1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput. Molecular & Cellular Proteomics 9:1400 -1410, 2010.Oxidation of cysteine residues plays a critical role in modifying the structure and function of many proteins. Although cysteine oxidation is a tightly regulated biological process, nonenzymatic processes can contribute substantially to its levels, such as during oxidative stress. Regulatory oxidation states such as disulfide bonding and S-nitrosylation are readily modulated (1) and play an essential role in many physiological processes, including cell cycle, growth, death, and differentiation (2). In contrast, prolonged accumulation of reactive oxygen species is associated with many pathological conditions and leads to stable overoxidized states (sulfinic and sulfonic acid) that may disrupt redox regulation and protein function (3) and, in most cases, are thought to be nonregenerative.Assays capable of comprehensively assessing the dynamic changes in site-specific oxidation states are especially critical to understanding the contribution of redox status to many diseases. Numerous redox-sensitive proteins, including essential cellular regulators such as p53, have been described previously (for review, see ref. 4). However, technical factors have hampered the identification of specific site(s) of modification and characterization of their redox status in cells. Sitedirected mutagenesis is often employed to determine whether specific cysteines have redox-regulated functional roles (1), but this approach provides no information on the oxidation status of the endogenous protein. In addition, cysteine oxidation is dynamically dependent on the concentration, location, and specificity of small-molecule oxidants (5) and regulators of various antioxidant enzymes (6). Thiol pKa (7), solvent accessibility, and ...

Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

“…Held et al [16] used a differential alkylation approach, along with proteolysis and multiple reaction monitoring (MRM) peptide mass spectrometry, to identify redox sensitive cysteines in endogenous p53. Only seven of the 10 Cys residues could be monitored using this approach (all but Cys229, 238, and 242).…”

“…There is clear and mounting evidence suggesting that redox-regulation of specific Cys residues in p53 plays a crucial role in controlling its activity. The redox-state of p53 cysteine residues influence its capacity to bind to consensus DNA in vitro and in vivo [8][9][10][11][12][13] and oxidation of endogenous p53 cysteines has been observed in both stressed and unstressed cells [14][15][16]. Proteins responsible for modulating Cys redox-state, such as thioredoxin (Trx) and redox factor 1 (ref- 1), participate in the regulation of p53 activity [17,18], and multiple signaling pathways exist that link reactive oxygen species (ROS) and reactive nitrogen species (RNS) with p53 [19,20].…”

Applications of targeted proteomics in systems biology and translational medicine

“…Danielson and coworkers used antibodies specific for 3‐nitrotyrosine to quantify the levels of this modification in alpha‐synuclein residues 62. Held and coworkers developed a new method for studying oxidation in response to reactive oxygen species, termed oxMRM 63. In a tour‐de‐force application of their method, they examined site‐specific cysteine oxidation status of endogenous p53, finding that residue C182 at the dimerization interface of the DNA‐binding domain is susceptible to diamide oxidation.…”

Paulownia tomentosa (Princess Tree) Extract Reduces DNA Damage and Induces DNA Repair Processes in Skin Cells

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“…The general response to oxidative stress is cell cycle arrest through the expression of various inhibitor proteins (such as p21). Nevertheless, ROS also serve useful roles within the body including intracellular and intercellular communication [44].…”