Laser ablation ICP-MS (LA-ICP-MS) has recently been applied to the determination of protein-bound metals using gels and gel blots. As all structural information is lost in the plasma flame, a direct determination of specific proteins is not possible. To overcome this problem, a novel application of LA-ICP-MS is presented, which takes advantage of the well established Western-Blotting-Technique to specifically detect proteins by the use of antibodies conjugated to gold clusters. After protein separation by regular 1D SDS-PAGE and transfer to a blotting membrane, the protein was marked by subsequent use of a primary antibody raised in rabbit and a mouse-anti-rabbit antibody covalently coupled to a gold cluster. We showed that the gold cluster could be detected by LA-ICP-MS. In comparison to single atoms, metal clusters increase the sensitivity in correlation to the number of metal atoms in the cluster. Quantification of the labelled antibodies was carried out by calibration using matrix matched standards. By using LA-ICP-MS, a detection limit of about 0.20 amol for gold cluster labelled antibody as well as excellent linearity was reached. As LA-ICP-MS allows the simultaneous multielement determination of protein-bound metal(loid)s this method is well suited to studies that require both the simultaneous quantification of a specific protein and the protein-bound metal(loid)s. This method is suitable for any protein for which a primary antibody is available. Here we applied it to analysis of the Mre11 protein in crude lysates of CHO-K1 fibroblasts. This protein is essential for mediating genome stability in mammalian cells and involved in DNA-repair and recombination.

Bone cells contacting nickel (Ni)-containing implant materials may be affected by Ni species via disturbed signaling pathways involved in bone cell development. Here we analyze effects of the Ni-containing steel 316L and major metal constituents thereof on bone morphogenetic protein-2 (BMP-2)-induced alkaline phosphatase (ALP) of MC3T3-E1 cells. While cells grew normally on 316L, cellular Ni content increased 10-fold vs. control within 4 days. With respect to the major components of 316L, Ni2+ (3-50 microM) was most inhibitory to BMP-2-induced ALP, whereas even 50 microM Fe3+, Cr3+, Mo5+, or Mn2+ had no such effect. In line with this, BMP-2-induced ALP was significantly reduced in cells on 316L. This effect was not prevented by the metal ion chelator diethylenetriaminepentaacetic acid (DTPA). Instead, DTPA abolished the stimulatory effect of BMP-2 on ALP, pointing to chelatable metal ions involved. Zn2+, as one possible candidate, antagonized the Ni2+ inhibition of BMP-2-induced ALP in both MC3T3-E1 and human bone marrow stromal cells. Results show that cells contacting 316L steel are exposed to increased concentrations of Ni which suffice to impair BMP-2-induced ALP activity. Zn2+, as a competitor of this inhibition, may help to restore normal osteoblastic function and bone development under these conditions.