Remarkable effect of mobile phasebuffer on the SEC-ICP-AES derived Cu, Fe and Zn-metalloproteome pattern of rabbit blood plasma

Abstract

The development of an analytical method to quantify the major Cu, Fe and Zn-containing metalloproteins in mammalian plasma has been recently reported. This method is based on the separation of plasma proteins by size exclusion chromatography (SEC) followed by the on-line detection of the metalloproteins by an inductively coupled plasma atomic emission spectrometer (ICP-AES). To assess whether the mobile phase buffer can affect the SEC-ICP-AES-derived metalloproteome pattern, thawed rabbit plasma was analyzed using phosphate buffered saline (PBS)-buffer (0.15 M, pH 7.4), Tris-buffer (0.1 and 0.05 M, pH 7.4), Hepes-buffer (0.1 M, pH 7.4) or Mops-buffer (0.1 M, pH 7.4). In contrast to the Cu-specific chromatograms, the Fe and Zn-specific chromatograms that were obtained with Tris, Hepes and Mops-buffer were considerably different from those attained with PBS-buffer. The Tris, Hepes and Mops-buffer mediated redistribution of ∼25% plasma Zn²⁺ from <100 kDa to >100–600 kDa plasma proteins and to a smaller extent to a <10 kDa (Tris)₂Zn²⁺-complex can be rationalized in terms of the abstraction of Zn²⁺ from the weak binding site on albumin. In contrast, only Hepes and Mops-buffer redistributed ∼20% of plasma Fe³⁺ from the <100 kDa to the >600 kDa elution range. Based on these results and considering that the utilization of PBS-buffer has previously resulted in the detection of a number of Cu, Fe and Zn-containing metalloentities in rabbit plasma that was most consistent with literature data, this mobile phase buffer is recommended for metallomic studies regarding mammalian blood plasma.