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Identification of mouse brain proteins associated with isoform 3 of metallothionein

¹ Department of Biochemistry, Molecular Biology, and Biophysics, and ² Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455, USA

(RECEIVED October 21, 2004; FINAL REVISION January 11, 2005; ACCEPTED January 14, 2005)

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.

Keywords: metallothionein-3; partner proteins; mass spectrometry; proteomics; mouse brain

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041113005.

Reprint requests to: Bruce L. Martin or Ian M. Armitage, Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street, SE, Minneapolis, MN 55455, USA; e-mail: marti285{at}umn.edu or armitage{at}msi.umn.edu; fax: (612) 625-2163.

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