Use of automated smoothing and deconvolution procedures for the determination of human breast cancer estrogen and progesterone receptor isoforms, after high-performance size-exclusion chromatography

Abstract

Automated smoothing and deconvolution procedures were used to analyse complex chromatographic patterns of human breast cancer estrogen and progesterone receptors, obtained by size-exclusion chromatography. By injecting, first, different known amounts of a radio-iodinated protein in a TSK G-3000 SW column, constructed complex chromatograms (twelve chromatograms) with known peak positions were obtained, and were further treated by mathematical functions to determine a smooth-deconvolution strategy, which could be used with unknown chromatographic patterns. The determination of peak areas by a “curve-fit” program showed a good correlation with the amounts of protein injected ( r = 0.91). Human breast cancer estrogen receptors (56 cases) and progesterone receptors (45 cases) were chromatographed in a TSK G-3000 SW column, and further analysed with the smoothing and deconvolution procedures: eight different estrogen receptor isoforms were detected with molecular masses ranging from 530 000 [Stoke's radius ( R s) = 7.7 nm] to 23 000 ( R s = 2.6 nm), and eight progesterone receptor isoforms were observed with masses ranging from 680 000 ( R s = 8.6 nm) to 50 000 ( R s = 3.1 nm). The dissociation effect of KCl (0 to 1 M) on receptor structure yielded different proportions of receptor isoforms, but did not modify their different peak positions.