Quantification of Trace Elements in Protein Bands Using Synchrotron Radiation X-ray Fluorescence after Electrophoretic Separation

A procedure was proposed for quantification of Fe, Cu and Zn in protein bands using synchrotron radiation X-ray fluorescence (SRXRF) after isoelectric focusing (IEF). Calibration standards were prepared by adding certain amounts of metal ions and free-metal proteins to electrophoresis gel. The calibration curves were obtained in the range of 0–8 μmg/g metals, and linear relationship was observed between dosage of metals and fluorescent intensity (R2 > 0.99). The detection limits were 2.43, 1.12, and 0.96 μmg/g for Fe, Cu and Zn, respectively, by the proposed method. Recoveries of 90.4% and 115.7% for Fe and Zn, respectively, were obtained when applying the procedure to quantification of the trace elements in protein bands of human hemoglobin separated with IEF. The combined techniques of SRXRF and IEF have advantages of quantifying trace elements in proteins and identifying proteins simultaneously. It can be employed for speciation analysis of trace elements in biological samples.