Imaging Metals in Proteins by Combining Electrophoresis with Rapid X-ray Fluorescence Mapping

Abstract

Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal-protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.

Calcium-dependent copper redistributions in neuronal cells revealed by a fluorescent copper sensor and X-ray fluorescence microscopy

“…XRFM affords, without any added reagents, a direct method for measuring total copper and other element distributions by their synchotroninduced X-ray fluorescence signatures (29,(37)(38)(39)(40). In particular, the instrument at the 2-ID-E beamline at the Advanced Photon Source of the Argonne National Laboratory boasts a spatial resolution of 200 nm, which makes it appropriate for examining the subcellular elemental distributions of single cells (41).…”

XFM of “Trace Metals” in Cultured Cells: Framing the Picture

“…Using other techniques, such as immunofluorescence, we can provide a biological context for the metal translocation observed by XFM analysis. As we continue to explore the translocation of metals in cells, we will not only utilize cryofixation and current biochemical techniques, but will continue to pursue developing and newly developed x-ray fluorescence techniques such as the bionanoprobe and a new technique, that we have recently published, combining 2D electrophoresis with XRF detection methods [9].…”

Synchrotron Radiation Spectroscopic Techniques as Tools for the Medicinal Chemist: Microprobe X-Ray Fluorescence Imaging, X-Ray Absorption Spectroscopy, and Infrared Microspectroscopy

“…[58] XAS is beneficial for the study of bioinorganic systems such as metal-and metalloid-containing drugs and enzymes since it is an extremely versatile and highly sensitive technique capable of providing information regarding metals in many physical states and chemical environments. [58][59][60] The technique can be applied to bulk or microprobe analyses of samples which include: solids, biological solutions, electrophoresis gels, and healthy or diseased mammalian cells and tissue. [58][59][60] XAS, in contrast to XRF spectroscopy, cannot feasibly be performed using a laboratory X-ray source.…”

“…[58][59][60] The technique can be applied to bulk or microprobe analyses of samples which include: solids, biological solutions, electrophoresis gels, and healthy or diseased mammalian cells and tissue. [58][59][60] XAS, in contrast to XRF spectroscopy, cannot feasibly be performed using a laboratory X-ray source. Instead the highly intense and tunable X-rays generated at a synchrotron are essential for XAS.…”