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Quantitation of the dynamic profiles of the innate immune response using multiplex selected reaction monitoring-mass spectrometry.

ABSTRACT: 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.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC3675810 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Quantitation of the dynamic profiles of the innate immune response using multiplex selected reaction monitoring-mass spectrometry.

Zhao Yingxin Y Tian Bing B Edeh Chukwudi B CB Brasier Allan R AR

Molecular & cellular proteomics : MCP 20130215 6

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 intrac ...[more]

PMID: 23418394

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Project description:For many research questions in modern molecular and systems biology, information about absolute protein quantities is imperative. This information includes, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes, or quantitative comparisons of different proteins within one sample or across samples. To date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers of samples. Here we describe and demonstrate the utility of a targeting MS technique for the estimation of absolute protein abundance in unlabeled and nonfractionated cell lysates. The method is based on selected reaction monitoring (SRM) mass spectrometry and the "best flyer" hypothesis, which assumes that the specific MS signal intensity of the most intense tryptic peptides per protein is approximately constant throughout a whole proteome. SRM-targeted best flyer peptides were selected for each protein from the peptide precursor ion signal intensities from directed MS data. The most intense transitions per peptide were selected from full MS/MS scans of crude synthetic analogs. We used Monte Carlo cross-validation to systematically investigate the accuracy of the technique as a function of the number of measured best flyer peptides and the number of SRM transitions per peptide. We found that a linear model based on the two most intense transitions of the three best flying peptides per proteins (TopPep3/TopTra2) generated optimal results with a cross-correlated mean fold error of 1.8 and a squared Pearson coefficient R(2) of 0.88. Applying the optimized model to lysates of the microbe Leptospira interrogans, we detected significant protein abundance changes of 39 target proteins upon antibiotic treatment, which correlate well with literature values. The described method is generally applicable and exploits the inherent performance advantages of SRM, such as high sensitivity, selectivity, reproducibility, and dynamic range, and estimates absolute protein concentrations of selected proteins at minimized costs.

Dataset Information

Quantitation of the dynamic profiles of the innate immune response using multiplex selected reaction monitoring-mass spectrometry.

Publications

Quantitation of the dynamic profiles of the innate immune response using multiplex selected reaction monitoring-mass spectrometry.

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