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Molecular & Cellular Proteomics 4:827-834, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

A High Throughput Method for the Detection of Metalloproteins on a Microgram Scale^*

Martin Högbom^,, Ulrika B. Ericsson^¶, Robert Lam^||, M. Amin Bakali H.^¶, Ekaterina Kuznetsova^**, Pär Nordlund^¶ and Deborah B. Zamble^,

From the Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 596, SE-75124 Uppsala, Sweden, the ^¶ Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden, ^|| Affinium Pharmaceuticals, 100 University Avenue, 10th Floor, South Tower, Toronto, Ontario M5J 1V6, Canada, the ^** Banting and Best Department of Medical Research, University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada, and the Department of Chemistry, Lash Miller Chemical Laboratories, University of Toronto, 80 St. George St., Toronto, Ontario M5S 3H6, Canada

Proteins that bind transition metals make up a substantial portion of the proteome, and the identification of a metal cofactor in a protein can greatly facilitate its functional assignment and help place it in the context of known cellular pathways. Existing methods for the detection of metalloproteins generally consume large amounts of protein, require expensive equipment, or are very labor intensive, rendering them unsuitable for use in high throughput proteomic initiatives. Here we present a method for the identification of metalloproteins that contain iron, copper, manganese, cobalt, nickel, and/or zinc that is sensitive, quick, robust, inexpensive, and can be performed with standard laboratory equipment. The assay is based on a combination of chemiluminescence and colorimetric detection methods, it typically consumes only 10 µg of protein, and most common chemical components of protein solutions do not interfere with metal detection. Analysis of 52 protein samples was compared with the results from inductively coupled plasma-atomic emission spectrometry to verify the accuracy and sensitivity of the method. The assay is conducted in a 384-well format and requires about 3 h for completion, including a 2-h wait; so whole proteomes can be assayed for metal content in a matter of days.

To whom correspondence may be addressed. Tel./Fax: 416-978-3568; E-mail: dzamble{at}chem.utoronto.ca

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