Stoichiometry and Absolute Quantification of Proteins with Mass Spectrometry Using Fluorescent and Isotope-labeled Concatenated

We have explored a general approach for the determina-tion of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spec-trometry. We engineered a gene to express green fluores-cent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB * contain concatenated se-quences of specific proteotypic peptides that are derived from the!, ", and # protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments