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

Yingxin Zhao, Bing Tian, Chukwudi B. Edeh, Allan R. Brasier

Research output: Contribution to journalArticlepeer-review

30 Scopus citations


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-I-κ Bα negative feedback loop indicate that about 20% of I-κBα 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-κBα 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 knockdownsuggests that a low nuclear threshold of NF-B 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.

Original languageEnglish (US)
Pages (from-to)1513-1529
Number of pages17
JournalMolecular and Cellular Proteomics
Issue number6
StatePublished - Jun 2013

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology


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