Abstract
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 microg 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.
Highlights
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
Metal Assay—To detect transition metals bound to proteins, a set of sequential tests was developed for analysis of samples in 384-well plates
Some of the common drawbacks of existing methods include the consumption of large amounts of protein, the complexity of the protocols, and specialized and expensive equipment [12,13,14,15]
Summary
Chemicals—Luminol, PAR, urea, bovine carbonic anhydrase, bovine copper-zinc superoxide dismutase, bovine heart cytochrome c, and all metal salts were purchased from Sigma. The buffer solutions were treated with Chelex-100 (Bio-Rad) to minimize trace metal contamination. Metal Analysis—Urea (4 l of an 8 M solution) was added to the wells of a non-transparent, 384-well plate with optical bottom (Nunc). One protein (10 g) was added to each well in no more than 2 l of solution followed by the addition of 10 l of a freshly prepared luminol solution (11 mM luminol, 500 mM Na2CO3, 230 mM H2O2) to all the wells. The samples were diluted to ϳ10 M in Chelex-treated 20 mM HEPES, pH 7.5, and 50 mM NaCl. For some samples 5% glycerol (v/v) was used to minimize precipitation; our previous studies of standard metal solutions demonstrated that this concentration of glycerol did not affect quantitation by ICP-AES [16]
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