Detection of phosphorylated subunits by combined LA-ICP-MS and MALDI-FTICR-MS analysis in yeast mitochondrial membrane complexes separated by blue native/SDS-PAGE

We report on the identification of phosphorylated subunits of yeast mitochondrial ATPase using a novel screening technique in combination with BN/SDS-PAGE. Protein complexes present in yeast mitochondrial membranes were separated in their native state in the first dimension and their subunit composition was resolved by SDS-PAGE in the second dimension. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to rapidly screen for the presence of phosphorus in the subunits. The detection limits of elements investigated in selected protein spots are in the low mu g g(-1) concentration range. Sulfur was used as the internal standard element for quantification. Phosphorus was detected in two of the proteins, that were identified by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) as subunits Atp1p and Atp2p of the ATPase. These results were confirmed by Western blot analysis using antibodies directed against phosphorylated amino acids. The combination of LA-ICP-MS and MALDI-FrICR-MS with BN/SDS-PAGE provides a fast and sensitive tool for structure analysis of phosphorus and metal-containing subunits of membrane protein complexes. (c) 2005 Elsevier B.V. All rights reserved.