Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and...

Knowledge on absolute protein concentrations is mandatory for the simulation of biological processes in the context of systems biology. A novel approach for the absolute quantification of proteins at a global scale has been developed and its applicability demonstrated using glucose starvation of the Gram-positive model bacterium Bacillus subtilis and the pathogen Staphylococcus aureus as proof-of-principle examples. Absolute intracellular protein concentrations were initially determined for a preselected set of anchor proteins by employing a targeted mass spectrometric method and isotopically labeled internal standard peptides. Known concentrations of these anchor proteins were then used to calibrate two-dimensional (2-D) gels allowing the calculation of absolute abundance of all detectable proteins on the 2-D gels. Using this approach, concentrations of the majority of metabolic enzymes were determined, and thus a quantification of the players of metabolism was achieved. This new strategy is fast, cost-effective, applicable to any cell type, and thus of value for a broad community of laboratories with experience in 2-D gel-based proteomics and interest in quantitative approaches. Particularly, this approach could also be utilized to quantify existing data sets with the aid of a few standard anchor proteins.