Absolute Quantification and Stoichiometry Determination of Protein Complexes; A Novel and Cost-efficient Method to Produce Exact Equimolar Mixture of Standard Peptides

摘要

>RP-125Determination of protein complex stoichiometry can be achieved by absolute quantification of the interacting subunits 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 peptides mixture. Here we describe a novel and cost-efficient method to generate such an equimolar mixture of internal standard peptides applicable to absolute quantification of proteins and subsequent stoichiometry determination of complex constituents. We call this method the Equimolarity through Equalizer Peptide (EtEP) strategy. All selected internal standard peptides are chemically synthesized concatenated to a N-terminal equalizer peptide, which is a short peptide with a high ionization efficiency and an optimized trypsin cleavage site. Trypsinization of the concatamers releases exact equimolar amount of standard peptides and the equalizer peptide. By normalizing all internal standard peptides to the signal generated by the equalizer peptide an equimolar mixture of standard peptides of high accuracy can be generated and absolute quantification of any protein of interest can be achievable via the equalizer peptide. Hence, the equalizer peptide is the only peptide species from which the exact amount has to be determined by amino acid analysis. Additionally, to cut the costs for quantification, we make use of the mTRAQ reagent to introduce the isotopic label. 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. In summary the EtEP strategy allows the generation of an equimolar mixture of standard peptides and significantly decreases the costs of absolute quantification and is ideally suited for a higher throughput in stoichiometry determination of protein complexes.