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-κB and IRF3 signaling arms, with IRF3 demonstrating a transient response, whereas NF-κB underwent a delayed secondary amplification phase. Our measurements of the NF-κB-IκBα negative feedback loop indicate that about 20% of IκBα 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 IκBα 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-κB is required for inducing target gene expression, and that there is cross-inhibition of the NF-κB and IRF3 signaling arms. Finally, we were able to measure delayed noncanonical NF-κB activation by quantifying the abundance of the processed (52 kDa) NF-κB2 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.
Highlights
From the ‡Institute for Translational Sciences, University of Texas Medical Branch, Galveston, Texas 77555; §Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555; ¶Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555
Development of selected reaction monitoring (SRM) Assays for Major Regulatory Components of innate immune response (IIR)—In this study, we focused on understanding the kinetics of the two nuclear factor (NF)-B pathways and the RIG-I-IRF3 pathway, the major signaling effectors of the IIR
We previously developed a workflow for selecting highresponding signature peptides of low-abundance proteins
Summary
Reagents and Chemicals—All reagents were American Chemical Society grade or higher. All solvents used, including water, methanol, and acetonitrile (ACN), were LC/MS grade. The remaining peptides were considered as signature peptide candidates and chemically synthesized in stable isotope labeled, crude, and unpurified forms for selecting SRM precursor-product ion (Q1/Q3) transitions and evaluating SRM assay specificity. The analyte peptides and their stable isotopically labeled peptide standard (SIS) analogs that had same chromatographic retention times (variance below 0.05 min) and relative product ion intensities (Ϯ20% variance in the relative ratios for each fragment) were considered as high-specificity signature peptides that were free of matrix interference from co-eluting ion. These peptides were chosen as the high-responding signature peptides of the target protein. The native versus SIS peptide ratios measured in the two replicate LC-SRM-MS analyses were averaged, and the mean and standard deviation were calculated
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