title: Stoichiometry determination of the MP1-p14 complex using a novel and cost-efficient method to produce an equimolar mixture of standard peptides.
abstract: Determination of protein complex stoichiometry can be achieved by absolute quantification of the interacting constituents based on isotope dilution mass spectrometry. Current available platforms for the generation of standard peptides are cost-intensive and deliver variable results concerning the equimolarity of the standard peptides. Here we describe a novel and cost-efficient method to generate an equimolar mixture of standard peptides, which we call the equimolarity through equalizer peptide (EtEP) strategy. The rationale of the strategy allows equalization of internal standard peptides and absolute quantification of any protein of interest via a single peptide, from which the exact amount was determined by amino acid analysis. This and the use of the mTRAQ reagent significantly decrease the costs of absolute quantification and stoichiometry determination. We used the EtEP strategy to determine the MP1-p14 complex stoichiometry of two different concentrations, one simulating a condition following tandem affinity purification. Absolute quantification of MP1-p14 was performed on two different mass spectrometers, and the 1:1 stoichiometry was confirmed with high accuracy and precision. On the basis of the quantification of MP1-p14, we demonstrate the importance to assess completeness of protein digestion and discuss the use of peptides containing labile amino acids and the choice of instrumentation.
authors.affiliation: Research Institute of Molecular Pathology, 1030 Vienna, Austria. johann.holzmann@imp.ac.at
authors.forename: Johann
authors.initials: J
authors.lastname: Holzmann
authors.forename: Peter
authors.initials: P
authors.lastname: Pichler
authors.forename: Mathias
authors.initials: M
authors.lastname: Madalinski
authors.forename: Robert
authors.initials: R
authors.lastname: Kurzbauer
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D010455:Peptides / Q000032:analysis* / Q000138:chemical synthesis; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry / Q000191:economics* / Q000295:instrumentation / Q000379:methods*
chemical_list: D000596:Amino Acids; C426009:LAMTOR2 protein, mouse; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins