The identification and quantification of metal atoms in proteins using microPIXE: A critical evaluation

Abstract

Around one third of all known protein molecules contain a small number of metal atoms which are often crucial to the biological function of the molecule. Identifying and quantifying these is not possible using commonly available analysis tools and there is a growing awareness that a majority of the metal atoms in the over 38,000 metalloprotein structures deposited in the Protein Data Bank are misidentified, with major consequences for the interpretation of protein function and mechanism. In the 1990s the authors developed a method for the identification and quantification of metals in liquid and crystalline protein samples by combining microbeam Proton Induced X-ray Emission and Rutherford backscattering. This has been used on overexpressed and purified proteins and has addressed many critical metal identification problems in structural biology. The capability has recently been extended to provide high throughput capability.In this paper we give an overview of the methodology and provide a critical evaluation of accuracy and detection limit and provide guidance on the optimisation of sample preparation and analysis conditions for these measurements. The discussion will be illustrated using some recent results illustrating the power of this method to answer biological questions.