OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum–protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum–protein bond stability under the conditions studied. SDS-PAGE in the absence of β-mercaptoethanol or dithiotreitol preserved the platinum–protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3–2.0 μg were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated.