title: A synthetic protein approach toward accurate mass spectrometric quantification of component stoichiometry of multiprotein complexes.
abstract: Quantitative description of protein interactions is crucial to understand and model molecular systems regulating various cellular activities. Here, we developed a novel peptide-concatenated standard (PCS) strategy for accurate mass spectrometric quantification of component stoichiometry of multiprotein complexes. In this strategy, tryptic peptides suitable for quantification are selected with their natural flanking sequences from each component of multiprotein complex and concatenated into a single synthetic protein called PCS. The concatenation guarantees equimolarity among the peptides added to the sample to obviate the need for preparation of accurately known amounts of individual peptides. The flanking sequences would equalize the excision efficiency of each peptide between the PCS and the target protein to improve the accuracy of quantification. To validate this strategy, we quantified the budding yeast eIF2Bgamma, the gamma subunit of eukaryotic initiation factor 2B, using a PCS composed of tryptic peptides from eIF2Bgamma with their flanking sequences. An identical sample-to-standard signal ratio was obtained within 5% measured error for these peptides, including the one prone to incomplete digestion, thereby proving the principle of PCS strategy. We applied the strategy to reveal the stoichiometry of the eIF2B-eIF2 complex using a PCS covering the 5 eIF2B and 3 eIF2 components. While the complex contained equimolar amounts of the eIF2B subunits, the ratio of each eIF2 subunit to eIF2B was 30-40%. The PCS strategy would provide a versatile method to quantitatively analyze compositional alteration of multiprotein complexes or dynamics of protein-protein interactions in response to various stimuli.
authors.affiliation: Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8561, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Kito
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Ota
authors.forename: Tomoko
authors.initials: T
authors.lastname: Fujita
authors.forename: Takashi
authors.initials: T
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000302:isolation & purification; D046911:Macromolecular Substances / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D014357:Trypsin
chemical_list: D002147:Calmodulin; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin