Structural identification and quantification of protein phosphorylations after gel electrophoretic separation using Fourier transform ion cyclotron resonance mass spectrometry and laser ablation inductively coupled plasma mass spectrometry

Abstract

In the present work mass spectrometric approaches are described for the identification of phosphorylated protein structures, and the direct quantification of protein–phosphorus contents, using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). An ultrahigh resolution FT-ICR-MS method was developed and applied for the structural identification of phosphorylations in proteins, using direct peptide mapping analysis with high mass accuracy of tryptic phosphorylated fragments. The application of this method to human tau proteins, one of the key proteins for the formation of neurofibrillary tangles in Alzheimer’s disease, provided the identification of 17 phosphorylation sites. A high-sensitivity inorganic mass spectrometric technique has been developed for the direct determination of phosphor and sulfur concentrations in proteins separated by two-dimensional (2D) gel electrophoresis. Quantitative P and S determination in protein gel spots was performed with an optimized method using a double-focusing sector field ICP mass spectrometer coupled to a laser ablation chamber (LA-ICP-MS). Two different quantification strategies were applied: (i) determination of P and S in gel spots by LA-ICP-MS, following the determination of these elements in blank gel after trypsin and HNO 3 digestion using ICP-SFMS; (ii) a new quantification procedure by LA-ICP-MS was developed for the direct microlocal analysis in small protein spots from 2D gels. A solution-based calibration strategy in LA-ICP-MS was proposed for the quantification procedure using an ultrasonic nebulizer for introduction of calibration standard solutions coupled to the laser ablation chamber. Cobalt was used as an internal standard element, and was added to the gel at a defined concentration. The quality of phosphor determination by LA-ICP-MS was ascertained with β-casein as reference material. In a first application to the multi-phosphorylated tau protein, an average phosphorus content of ca. 20% was determined. The present results demonstrate the analytical merit of the combination of high resolution FT-ICR-MS and LA-ICP-MS for the molecular characterization of phosphorylated protein structures and determination of phosphorus and sulfur from 2D gels.