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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.

SUBMITTER: Held JM

PROVIDER: S-EPMC2938085 | biostudies-other | 2010 Jul

REPOSITORIES: biostudies-other

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Targeted quantitation of site-specific cysteine oxidation in endogenous proteins using a differential alkylation and multiple reaction monitoring mass spectrometry approach.

Held Jason M JM Danielson Steven R SR Behring Jessica B JB Atsriku Christian C Britton David J DJ Puckett Rachel L RL Schilling Birgit B Campisi Judith J Benz Christopher C CC Gibson Bradford W BW

Molecular & cellular proteomics : MCP 20100316 7

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 alk ...[more]

PMID: 20233844

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Project description:The innate immune response (IIR) is a coordinated intracellular signaling network activated by the presence of pathogen-associated molecular patterns that limits pathogen spread and induces adaptive immunity. Although the precise temporal activation of the various arms of the IIR is a critical factor in the outcome of a disease, currently there are no quantitative multiplex methods for its measurement. In this study, we investigate the temporal activation pattern of the IIR in response to intracellular double-stranded RNA stimulation using a quantitative 10-plex stable isotope dilution-selected reaction monitoring-MS assay. We were able to observe rapid activation of both NF-?B and IRF3 signaling arms, with IRF3 demonstrating a transient response, whereas NF-?B underwent a delayed secondary amplification phase. Our measurements of the NF-?B-I?B? negative feedback loop indicate that about 20% of I?B? in the unstimulated cell is located within the nucleus and represents a population that is rapidly degraded in response to double-stranded RNA. Later in the time course of stimulation, the nuclear I?B? pool is repopulated first prior to its cytoplasmic accumulation. Examination of the IRF3 pathway components shows that double-stranded RNA induces initial consumption of the RIG-I PRR and the IRF3 kinase (TBK1). Stable isotope dilution-selected reaction monitoring-MS measurements after siRNA-mediated IRF3 or RelA knockdown suggests that a low nuclear threshold of NF-?B is required for inducing target gene expression, and that there is cross-inhibition of the NF-?B and IRF3 signaling arms. Finally, we were able to measure delayed noncanonical NF-?B activation by quantifying the abundance of the processed (52 kDa) NF-?B2 subunit in the nucleus. We conclude that quantitative proteomics measurement of the individual signaling arms of the IIR in response to system perturbations is significantly enabled by stable isotope dilution-selected reaction monitoring-MS-based quantification, and that this technique will reveal novel insights into the dynamics and connectivity of the IIR.

Dataset Information

Targeted quantitation of site-specific cysteine oxidation in endogenous proteins using a differential alkylation and multiple reaction monitoring mass spectrometry approach.

Publications

Targeted quantitation of site-specific cysteine oxidation in endogenous proteins using a differential alkylation and multiple reaction monitoring mass spectrometry approach.

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