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Analysis of transcription complexes and effects of ligands by microelectrospray ionization mass spectrometry

Nature Biotechnology volume 17, pages 1214–1218 (1999)Cite this article

Abstract

The human vitamin D receptor (VDR) and retinoid X receptor-α (RXRα) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in the presence of their known endogenous ligands 1α,25-dihydroxyvitamin D3 (1,25-[OH]2D3) and 9-cis-retinoic acid (9-c-RA). We used rapid buffer exchange gel filtration in conjunction with microelectrospray ionization mass spectrometry (μESI-MS) to study the binding of these receptors to the osteopontin vitamin D response element (OP VDRE). In the absence of DNA, both VDR and RXRα existed primarily as monomers, but in the presence of OP VDRE, homodimeric RXRα and heterodimeric RXRα–VDR complexes were shown to bind OP VDRE. Addition of 9-c-RA increased RXRα homodimer–OP VDRE complexes, and addition of 1,25-(OH) 2D3 resulted in formation of 1,25-(OH)2D 3–VDR–RXRα–OP VDRE complexes. Addition of low-affinity binding ligands had no detectable effect on the VDR–RXRα–OP VDRE transcription complex. These results demonstrate the utility of μESI-MS in analyzing multimeric, high-molecular-weight protein–protein and protein–DNA complexes, and the effects of ligands on these transcriptional complexes.

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Figure 1: Gel shift analyses of VDR and RXRα complexes with OP VDRE DNA.
Figure 2: Positive-ion μESI-MS of protein and protein–DNA mixtures, over mass range 3,000–5,000 amu, in 10 mM NH4HCO3 at pH 8.0.
Figure 3: Positive-ion μESI-MS of protein–DNA mixtures over mass range 3,500–4,100 amu in 10 mM NH4HCO3 at pH 8.0.
Figure 4: Positive-ion μESI-MS of protein–DNA–ligand mixtures over 3,400–4100 amu.

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Acknowledgements

We thank Mrs. Diana Ayerhart for her help in preparing this manuscript. Supported by NIH grant DK 25409 (R.K.) and Finnigan MAT#1 (S.N.)

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Authors and Affiliations

  1. Nephrology Research Unit, Mayo Clinic, Rochester, 55905, MN

    Theodore A. Craig & Rajiv Kumar

  2. Biomedical Mass Spectrometry Facility, Mayo Clinic, Rochester, 55905, MN

    Linda M. Benson, Andy J. Tomlinson & Stephen Naylor

  3. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, 55905, MN

    Linda M. Benson, Andy J. Tomlinson, Stephen Naylor & Rajiv Kumar

  4. Department of Medicine, Mayo Clinic, Rochester, 55905, MN

    Rajiv Kumar

  5. Department of Pharmacology and Clinical Pharmacology Unit, Mayo Clinic, Rochester, 55905, MN

    Stephen Naylor

  6. Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, 99352, WA

    Timothy D. Veenstra

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Correspondence to Stephen Naylor or Rajiv Kumar.

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Craig, T., Benson, L., Tomlinson, A. et al. Analysis of transcription complexes and effects of ligands by microelectrospray ionization mass spectrometry. Nat Biotechnol 17, 1214–1218 (1999). https://doi.org/10.1038/70767

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