Simultaneous Cu-, Fe-, and Zn-specific detection of metalloproteins contained in rabbit plasma by size-exclusion chromatography-inductively coupled plasma atomic emission spectroscopy

Analytical methods which are capable of determining the plasma or serum metalloproteome have inherent diagnostic value for human diseases associated with increased or decreased concentrations of specific plasma metalloproteins. We have therefore systematically developed a method to rapidly determine the major Cu-, Fe-, and Zn-containing metalloproteins in rabbit plasma (0.5 mL) based on size-exclusion chromatography (SEC; stationary phase Superdex 200, mobile phase phosphate-buffered saline pH 7.4) and the simultaneous online detection of Cu, Fe, and Zn in the column effluent by an inductively coupled plasma atomic emission spectrometer (ICP-AES). Whereas most previous studies reported on the analysis of serum, our investigations clearly demonstrated that the analysis of plasma within 30 min of collection results in the detection of one more Cu peak (blood coagulation factor V) than has been previously reported (transcuprein, ceruloplasmin, albumin-bound Cu, and small molecular weight Cu). The average amount of Cu associated with these five proteins corresponded to 21, 18, 21, 30 and 10% of total plasma Cu, respectively. In contrast, only two Fe metalloproteins (ferritin and transferrin, corresponding to an average of 9 and 91% of total plasma Fe) and approximately five Zn metalloproteins (alpha(2)-macroglobulin and albumin-bound Zn, which corresponded to an average of plasma Zn) were detected. Metalloproteins were assigned on the basis of the coelution of the corresponding metal and protein identified by immunoassays or activity-based enzyme assays. The SEC-ICP-AES approach developed allowed the determination of approximately 12 Cu, Fe, and Zn metalloproteins in rabbit plasma within approximately 24 min and can be applied to analyze human plasma, which is potentially useful for diagnosing Cu-, Fe-, and Zn-related diseases.