Mass Spectrometric Analysis of a Native Zinc-Finger Structure: The Glucocorticoid Receptor DNA Binding Domain

H. Ewa Witkowska, Cedric H.L. Shackleton, John Y. Kim, Karin Dahlman-Wright, Jan-Ake Gustafsson

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

The DNA binding domain of the glucocorticoid receptor (GR DBD, recombinant, human amino acids 419–501) was analyzed intact under neutral conditions by electrospray ionization mass spectrometry (ESI MS). The Zn-containing GR DBD and its Cd-containing counterpart both showed stoichiometry of two metal atoms attached to a molecule of metalloprotein (molecular mass 9600 ± 2.1 Da (n = 4) and molecular mass 9693 ± 1.3 Da, respectively). GR DBD analyzed at low pH gave the molecular mass expected for the apoprotein: 9474 ± 1 Da (n = 4) (average Mr9474.4). There was a difference in the distribution envelopes of molecular ions in ESI mass spectra of the Zn- and Cd-containing GR DBD's depending upon conditions of the ESI MS experiment. In acidic (denaturing) conditions, molecular ion envelopes moved toward lower m/z values, while at neutral pH in aqueous solvent, a characteristic low level of protonation was noted, the latter indicative of preservation of some higherorder structure during ESI MS analysis. For electrospray ionization mass spectrometric analysis, the native proteins in ammonium bicarbonate buffer were injected into a stream of 50 mM pyridine acetate (pH 5.9) and delivered to the VG BioQ instrument at a flow rate of 4 μL/min. Denatured proteins were analyzed either by injection into a stream of 50% acetonitrile/0.1% trifluoroacetic acid or on-line with HPLC using a reversed phase narrow bore PLRP-S column (1 x 50 mm).

Original languageEnglish (US)
Pages (from-to)3319-3324
Number of pages6
JournalJournal of the American Chemical Society
Volume117
Issue number12
DOIs
StatePublished - Jan 1 1995

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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