Detection of specific proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) using gold cluster labelled antibodies

Abstract

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.