Quantification of activated NF-κB/RelA complexes using ssDNA aptamer affinity - Stable isotope dilution - Selected reaction monitoring - Mass spectrometry

Yingxin Zhao, Steven Widen, Mohammad Jamaluddin, Bing Tian, Thomas Wood, Chukwudi B. Edeh, Allan R. Brasier

Research output: Contribution to journalArticle

32 Citations (Scopus)

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 KD 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.

Original languageEnglish (US)
JournalMolecular and Cellular Proteomics
Volume10
Issue number6
DOIs
StatePublished - Jun 2011

Fingerprint

Isotopes
Dilution
Mass spectrometry
Mass Spectrometry
Assays
Monitoring
Serine
Histone Acetyltransferases
Proteins
Trans-Activators
Eukaryotic Cells
Cytoplasm
Transcription Factors
Gene expression
Binding Sites
Cytokines
Gene Expression
Chemical activation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry
  • Medicine(all)

Cite this

Quantification of activated NF-κB/RelA complexes using ssDNA aptamer affinity - Stable isotope dilution - Selected reaction monitoring - Mass spectrometry. / Zhao, Yingxin; Widen, Steven; Jamaluddin, Mohammad; Tian, Bing; Wood, Thomas; Edeh, Chukwudi B.; Brasier, Allan R.

In: Molecular and Cellular Proteomics, Vol. 10, No. 6, 06.2011.

Research output: Contribution to journalArticle

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