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

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 g 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. Molecular & Cellular Proteomics 4:827-834, 2005.Proteomics is the biochemical and physiological characterization of all proteins and variants produced by a cell and includes determination of the structure and function of each protein, its interactions with other cellular factors, spatial and temporal location, and regulation (1, 2). This type of initiative is now possible on an unprecedented scale and motivates the challenge to develop technology and applications for systematic high throughput analysis (1, 3). One assay that would be a very useful addition to the existing battery of proteomic protocols is a quick and simple method for high throughput (HTP) 1 identification of transition metal-containing proteins. Current estimates suggest that metalloproteins make up about one-third of the proteome (4, 5), so the identification of a metal cofactor would facilitate the functional assignment of a protein and help place it in the context of known cellular pathways (6). Furthermore this information would be useful for selecting attractive targets for high throughput structural genomic efforts (3, 7) because metal cofactors can often be used to phase x-ray crystallographic data by anomalous dispersion methods, and the protein structure can be solved without the need for secondary experiments such as the production of selenomethionine-containing protein or heavy metal soaking of protein crystals (8 -11).The currently available methods for metal analysis of proteins are not suitable for routine HTP efforts. These techniques, such as atomic absorption spectrometry, inductively coupled plasma coupled with either atomic emission spectroscopy (ICP-AES) or mass spectrometry (ICP-MS), or synchrotron techniques such as x-ray fluorescence spectro...

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