title: Quantification of activated NF-kappaB/RelA complexes using ssDNA aptamer affinity-stable isotope dilution-selected reaction monitoring-mass spectrometry.
abstract: Nuclear Factor-κB (NF-κB) is a family of inducible transcription factors regulated by stimulus-induced protein interactions. In the cytoplasm, the NF-κB member RelA transactivator is inactivated by binding inhibitory IκBs, whereas in its activated state, the serine-phosphorylated protein binds the p300 histone acetyltransferase. Here we describe the isolation of a ssDNA aptamer (termed P028F4) that binds to the activated (IκBα-dissociated) form of RelA with a K(D) of 6.4 × 10(-10), and its application in an enrichment-mass spectrometric quantification assay. ssDNA P028F4 competes with cognate duplex high affinity NF-κB binding sites for RelA binding in vitro, binds activated RelA in eukaryotic nuclei and reduces TNFα-stimulated endogenous NF-κB dependent gene expression. Incorporation of P028F4 as an affinity isolation step enriches for serine 536 phosphorylated and p300 coactivator complexed RelA, simultaneously depleting IκBα·RelA complexes. A stable isotope dilution (SID)-selected reaction monitoring (SRM)- mass spectrometry (MS) assay for RelA was developed that produced a linear response over 1,000 fold dilution range of input protein and had a 200 amol lower limit of quantification. This multiplex SID-SRM-MS RelA assay was used to quantify activated endogenous RelA in cytokine-stimulated eukaryotic cells isolated by single-step P028F4 enrichment. The aptamer-SID-SRM-MS assay quantified the fraction of activated RelA in subcellular extracts, detecting the presence of a cytoplasmic RelA reservoir unresponsive to TNFα stimulation. We conclude that aptamer-SID-SRM-MS is a versatile tool for quantification of activated NF-κB/RelA and its associated complexes in response to pathway activation.
authors.affiliation: Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Yingxin
authors.initials: Y
authors.lastname: Zhao
authors.forename: Steven G
authors.initials: SG
authors.lastname: Widen
authors.forename: Mohammad
authors.initials: M
authors.lastname: Jamaluddin
authors.forename: Bing
authors.initials: B
authors.lastname: Tian
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Wood
authors.forename: Chukwudi B
authors.initials: CB
authors.lastname: Edeh
authors.forename: Allan R
authors.initials: AR
authors.lastname: Brasier
mesh_terms: D000595:Amino Acid Sequence; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive; D002138:Calibration; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004277:DNA, Single-Stranded / Q000737:chemistry*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D020672:I-kappa B Proteins / Q000737:chemistry; D007553:Isotope Labeling; D057230:Limit of Detection; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry; D016328:NF-kappa B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D053719:Tandem Mass Spectrometry / Q000379:methods; D051996:Transcription Factor RelA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology
chemical_list: D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D020672:I-kappa B Proteins; D046912:Multiprotein Complexes; D016328:NF-kappa B; C498143:RELA protein, human; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha