Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping.
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.
Finney
Lydia
L
Argonne National Laboratory, Illinois, USA. lfinney@anl.gov <lfinney@anl.gov>
Chishti
Yasmin
Y
Khare
Tripti
T
Giometti
Carol
C
Levina
Aviva
A
Lay
Peter A
PA
Vogt
Stefan
S
D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002857:Chromium / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D005453:Fluorescence; D007501:Iron / Q000032:analysis / Q000378:metabolism; D057230:Limit of Detection; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000032:analysis* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D020592:Shewanella / Q000737:chemistry / Q000378:metabolism; D014965:X-Rays
D001798:Blood Proteins; D008667:Metalloproteins; D008670:Metals; D011506:Proteins; D002857:Chromium; D007501:Iron; D010100:Oxygen