Mass spectrometric analysis of a native zinc-finger structure: The glucocorticoid receptor DNA binding domain

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

Research output: Contribution to journalArticlepeer-review


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 Mr 9474.4). 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 higher-order 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 × 50 mm).

Original languageEnglish (US)
JournalJournal of the American Chemical Society
Issue number12
StatePublished - Mar 29 1995

ASJC Scopus subject areas

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


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