MASS-SPECTROMETRIC ANALYSIS OF A NATIVE ZINC-FINGER STRUCTURE - THE GLUCOCORTICOID RECEPTOR DNA-BINDING DOMAIN

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 M(r) 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 mu L/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(l x 50 mm).