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

Abstract

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 μg 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–FTICR–MS with BN/SDS-PAGE provides a fast and sensitive tool for structure analysis of phosphorus and metal-containing subunits of membrane protein complexes.