The MIRAGE Method of Metalloproteomics

Metal ions play an important role in biological systems; approximately one third of all proteins have been estimated to contain a metal cofactor. In the past decades a number of breakthroughs in the research of metalloenzymes has been achieved, especially concerning the three dimensional structures of metalloproteins and functions of the metal sites. Nevertheless, it is still very difficult to comprehensively localize, identify and quantify metal–containing proteins in a particular organism. Because of the complex composition of biological samples, the use of high resolution separation techniques for proteins, in addition to high sensitivity metal analysis and quantification, is essential. Metal Isotope native RadioAutography in Gel Electrophoresis (MIRAGE) technology has been developed to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system. The procedure for MIRAGE involves four steps: (1) labeling of the target proteins with a suitable short–lived radioisotope (e.g. 56Mn, 59Fe, 64Cu, 65Ni, 69Zn, 99Mo, 187W); (2) separation of intact soluble holoproteins using native isoelectric focusing (IEF) combined with blue native polyacrylamide gel electrophoresis (BN–PAGE) into native–native 2D gel electrophoresis; (3) spot visualization and quantification using autoradiography; and (4) protein identification with tandem mass spectrometry. MIRAGE experiments were conducted on model microorganisms, wild–type Escherichia coli K12 and hyperthermophilic archaeon Pyrococcus furiosus, using metal isotopes of biological relevance (59Fe, 64Cu, 69Zn, 99Mo and 187W).BiotechnologyApplied Science