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SCMM investigators pioneer quantitative proteomics studies of the innate pathway

A series of papers reported by Dr. Zhao of the SCMM have illustrated the methodology for use of selected reaction monitoring (SRM) to profile the host cell stress response. Selected reaction monitoring (SRM) is a new ‘targeted’ MS approach for the detection and accurate quantification of a predetermined set of proteins in a complex background. In a SRM-MS assay, one or two signature proteotypic peptides unique to the protein of interest are selected to represent the protein. SRM-MS quantification of these signature peptides are performed on a triple quadrupole mass spectrometer (QQQ-MS), an instrument with the capability to selectively isolate precursor ions corresponding to the signature peptide m/z value and to monitor peptide-specific fragment ions that provide near-absolute structural specificity for the target protein. Using the technique of stable isotope dilution (SID), highly accurate measurements of the target protein can be measured. Dr. Zhao’s group developed a quantitative 10-plex SID-SRM assays to measure the kinetics of the human innate immune response (IIR) in epithelial cells.

The IIR is a coordinated intracellular signaling network activated by the pathogen-associated molecular patterns to limit pathogen spread and induce adaptive immunity. Dr. Zhao and collaborators observed that ds-RNA rapidly induces nuclear translocation of both NF-kB and IRF3 signaling arms; although IRF3 demonstrates a transient nuclear residence, NF-kB is controlled by a delayed secondary amplification phase. These measurements of the IkBa negative feedback regulator of NF-kB indicate that about 20% of IkBa in the unstimulated cell is located within the nucleus, and this fraction represents a population that is rapidly degraded in response to ds-RNA. Later in the time course of stimulation, the nuclear IkBa pool is repopulated first prior to its cytoplasmic accumulation. Examination of the IRF3 pathway components show that ds-RNA induces initial consumption of the RIG-I pattern recognition receptor and the IRF3 kinase complex. SID-SRM-MS measurements after siRNA- mediated IRF3 or RelA knockdown suggests that a low nuclear threshold of NF-kB is required for inducing target gene expression, and that there is cross-inhibition of the NF-kB and IRF3 signaling arms. Finally, we observe a delayed noncanonical NF-B activation by quantifying the abundance of the processed (52 kDa) NF-kB2 subunit in the nucleus. These studies indicate that quantitative proteomics measurement of the individual signaling arms of the IIR in response to system perturbations is significantly enabled by SID-SRM-MS-based quantification and that this technique will reveal novel insights into the dynamics and connectivity of the IIR.


  • Zhao, Y., Tian, B., Edeh, C.B., and Brasier, AR. Quantitation of the dynamic profiles of the innate immune response using multiplex selected reaction monitoring-mass spectrometry. Mol Cell Proteomics. 2013, 12: 1513-1529.
  • Zhao, Y, Widen, SG, Jamaluddin, M, Tian, B, Wood, TG and Brasier AR † Quantification of Activated NF-kB/RelA Complexes Using ssDNA Aptamer Affinity – Stable Isotope Dilution-Selective Reaction Monitoring-MS. Mol Cellular Proteomics, 2011, 10(6), 1-16. PMCID: PMC3108844.
  • Zhao, Y, and Brasier AR. Applications Of Selected Reaction Monitoring (SRM)-Mass Spectrometry (MS) For Quantitative Measurement Of Signaling Pathways. In "Quantitative Proteomics" Theme Issue. Methods, Elsevier Publishers, 2013 Feb 11. doi:pii: S1046-2023(13)00020-0. 10.1016/j.ymeth.2013.02.001.

SCMM investigators develop probabilistic method

Using Nuclear factor-kB (NF-kB) chromatin immunoprecipitation (ChIP)-next generation sequencing (Seq) data, SCMM investigators have developed and published a framework for iterative learning of regulatory networks. For every possible transcription factor binding site (TFBS)- putatively regulated gene pair, the relative distance and orientation are calculated to learn which TFBSs are most likely to regulate a given gene. Weighted TFBS contributions to putative gene regulation are integrated to derive an NF-κB gene network. A de novo motif enrichment analysis uncovers secondary TFBSs (AP1,SP1) at characteristic distances from NF-kB/RelA TFBSs. Comparison with experimental ENCODE ChIP-Seq data indicates that experimental TFBSs highly correlate with predicted sites. We observe that RelA-SP1 enriched promoters have distinct expression profiles from that of RelA-AP1, and are enriched in introns, CpG islands and DNase accessible sites. 17 novel NF-kB/RelA regulated genes were experimentally validated, including TANK, a negative feedback gene whose expression is NF-kB/RelA dependent and requires a functional interaction with the AP1 TFBSs. Our probabilistic method yields more accurate NF-kB/RelA -regulated networks than a traditional, distance-based approach, confirmed by both analysis of gene expression and increased informativity of Genome Ontology (GO) annotations. This analysis provides new insights how co-occurring TFBSs and local chromatin context orchestrate activation of NF-kB/RelA sub-pathways differing in biological function and temporal expression patterns.


  • Yang, J, Mitra, A, Dojer, N, Fu, S, Rowicka, M, and Brasier AR. A probabilistic approach to learning chromatin architecture and accurate inference of the NF-κB/RelA regulatory network using ChIP-Seq. Nuc Acids Res, 2013, in press.
  • Tian, B, Yang, J, and Brasier AR. Two-step Crosslinking for Analysis of Protein-Chromatin Interactions. Methods in Molecular Biology, Transcriptional Regulation, Methods and Protocols. A. Vercura, Editor. 2012; 809:105-20.

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