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

Abstract

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent 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 sequences 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. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits ␣, ␤, and ␥ was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies. Molecular & Cellular Proteomics 7:442-447, 2008.We describe a general method to determine the stoichiometry and absolute quantification of proteins, a method that substantively extends previously developed techniques. Many proteins assemble to form protein complexes to fulfill unique functional roles. Protein assemblies provide a functional diversity that can contextually change with time and space during a cellular lifecycle. Many of these complexes have been dissected and catalogued in different organisms using the combination of tandem affinity purification followed by identification of individual members by mass spectrometry (1, 2). Comprehensive identification of individual members in protein assemblies is now performed routinely using mass spectrometry (3, 4). However, the determination of stoichiometry and quantification of individual proteins in complexes or mixtures usually requires the use of analytical ultracentrifugation.Several techniques for protein quantification using mass spectrometry depend on quantification of peptides generated during their proteolytic digestion. Chemical labeling of peptides after digestion (5) or metabolic labeling of growing cells in the presence of labeled substrate are useful for relative or comparative analyses of different sample groups (6). It has been proposed that the absolute quantification of proteins in complex protein mixtures can be accomplished by isotope dilution using synthetic labeled internal standa...

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