title: Development of a generic approach to native metalloproteomics: application to the quantitative identification of soluble copper proteins in Escherichia coli.
abstract: A combination of techniques to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system has been developed. The procedure involves four steps: (1) labeling of the target proteins with a suitable short-lived radioisotope (suitable isotopes are (64)Cu, (67)Cu, (187)W, (99)Mo, (69)Zn, (56)Mn, (65)Ni); (2) separation of intact soluble holoproteins using native isoelectric focusing combined with blue native polyacrylamide gel electrophoresis into native-native 2D gel electrophoresis; (3) spot visualization and quantification using autoradiography; and (4) protein identification with tandem mass spectrometry. The method was applied to the identification of copper proteins from a soluble protein extract of wild-type Escherichia coli K12 using the radioisotope (64)Cu. The E. coli protein CueO, which has previously been only identified as a multicopper oxidase following homologous overexpression, was now directly detected as a copper protein against a wild-type background at an expression level of 0.007% of total soluble protein. The retention of the radioisotope by the copper proteins throughout the separation process corroborates the method to be genuinely native. The procedure developed here can be applied to cells of any origin, and to any metal having suitable radioisotopes. The finding that the periplasmic protein CueO is the only major form of soluble protein bound copper in E. coli strengthens the view that the bacterial periplasm contains only a few periplasmic copper proteins, and that the cytosol is devoid of copper proteins.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Ana-Maria
authors.initials: AM
authors.lastname: Sevcenco
authors.forename: Gerard C
authors.initials: GC
authors.lastname: Krijger
authors.forename: Martijn W H
authors.initials: MW
authors.lastname: Pinkse
authors.forename: Peter D E M
authors.initials: PD
authors.lastname: Verhaert
authors.forename: Wilfred R
authors.initials: WR
authors.lastname: Hagen
authors.forename: Peter-Leon
authors.initials: PL
authors.lastname: Hagedoorn
mesh_terms: D001400:Azurin / Q000737:chemistry / Q000378:metabolism; D003300:Copper / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D011868:Radioisotopes / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D008667:Metalloproteins; D008670:Metals; D011868:Radioisotopes; D001400:Azurin; D003300:Copper; D010088:Oxidoreductases; C499679:cueO protein, E coli