Analysis of proteins by particle induced X-ray emission

The development of a standard PIXE procedure dedicated to the metal analysis in proteins is of great interest for biochemists. In order to achieve this aim, the LIPSION microprobe facility at the Leipzig University was used with a 2.25 MeV scanning proton micro-beam for the investigation of several test systems with known metal concentrations and stoichiometries. STIM and RBS were performed in these systems to select the best technique for areal density determination. With this set-up STIM proved to be less sensitive to sample thickness inhomogeneities. Radiation damage was evaluated in real proteins, such as glyoxalase II and no effects were noticed for currents up to 500 pA. Glyoxalase II served also to demonstrate the ability of PIXE to determine the metal stoichiometry even for protein concentrations of ∼60 μM. The zinc phosphodiesterase and the flavorubredoxin lactamase domain were also analyzed. The use of PIXE, combined with STIM for areal density determination, turned out to be a very powerful technique in protein analysis, allowing for stoichiometry determinations of very small sample quantities without any further chemical preparation.