In-gel screening of phosphorus and copper, zinc and iron in proteins of yeast mitochondria by LA-ICP-MS and identification of phosphorylated protein structures by MALDI-FT-ICR-MS after separation with two-dimensional gel electrophoresis

Abstract

A new screening technique using two-dimensional gels was developed in order to rapidly identify various elements in well-separated protein spots. Yeast mitochondrial proteins were separated using two-dimensional gel electrophoresis (blue native/SDS 2D-PAGE) and marked by silver staining. The 2D gels were systematically analyzed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) using a double-focusing sector field instrument. From more than 60 mitochondrial protein spots in two-dimensional gels, phosphorus, sulfur and selected metals (Cu, Zn and Fe) were detected in a short analysis time by screening 2D gel with LA-ICP-MS using a focused laser beam. In selected protein spots a quantitative element determination was performed. Ion intensities of phosphorus and metals in single protein spots in the gels were measured at medium mass resolution using an optimized microanalytical method by LA-ICP-MS and in a solution of the gel (blank) after HNO3 digestion by ICP-MS. For quantification purposes sulfur was used as the internal standard element. The detection limits for phosphorus, sulfur, copper, zinc and iron in protein spots, determined in the gel blank (Coomassie staining), were 0.18 µg g−1, 1.3 mg g−1, 6.4 µg g−1, 17.6 µg g−1 and 9.5 µg g−1, respectively. In silver staining gel a detection limit for sulfur of 137 µg g−1 was measured. Matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) was applied for structure analysis and determination of phosphorylation sites of phosphorylated proteins. Results of the structure analysis of separated mitochondrial proteins obtained by MALDI-FTICR-MS were combined with those of the direct determination of phosphorus, sulfur and metal concentrations in protein spots in two-dimensional gels with LA-ICP-MS.