Elspeth F. Garman, Geoffrey W. Grime

The identification and quantification of metals bound to proteins is a crucial problem to be solved in structural biology. This paper describes the technique of particle induced X-ray emission with a microfocused beam (microPIXE) as a tool for analysing the elemental composition of liquid and crystalline protein samples. The proton beam induces characteristic X-ray emission from all elements in the protein, which can be interpreted in terms of the metal content of the protein molecule with a relative accuracy of between 10% and 20%. The compelling advantage of this method is that the sulphur atoms in the methionines and cysteines of the protein provide an internal calibration of the number of protein molecules present so that systematic errors are minimised and the technique is entirely internally self-consistent. This is achieved by the simultaneous measurement of the energy of backscattered protons (Rutherford backscattering), to enable us to determine the matrix composition and thickness, and so correct the PIXE data for the self-absorption of X-rays in the sample. The theoretical background to the technique is described, and the technical and experimental procedures are outlined. Examples of recent measurements are given which have informed a range of investigations in structural biology. The use of the technique is increasing and we envisage that future developments will enable it to become a routine high-throughput method.