Program

Oral Sessions

Day 4, May 18（Fri.）　15:45-16:05　Room B (Seiun 1)

4B-O2-P-1545　　PDF

High-throughput biosynthesis of stable isotope-labeled internal standards for global proteome quantification

(Ehime Univ.)
^oNobuaki Takemori

Absolute quantification of individual cellular proteins is necessary for constructing models to systematically understand biological processes. The most reliable quantitation of target proteins in crude biological samples is based on isotope dilution mass spectrometry, which requires precisely quantitated standard peptides. Today I will present an innovative approach, “MEERCAT” (Multiplexed Efficient Expression of Recombinant QconCATs), designed to create a large-scale standard peptide library for the absolute quantification of a cellular proteome via high-throughput synthesis of concatemer peptides, called QconCATs, in a wheat germ cell-free system. Wheat germ cell-free synthesis is a powerful in vitro translation system with which even artificial proteins such as QconCATs can be stably synthesized. Co-synthesis of multiple proteins, which is generally difficult with cell-based synthetic systems, is also possible. Using MEERCAT, we have succeeded in simultaneous synthesis of multiple QconCATs in quantities suitable for proteome quantification using mass spectrometry. For small-sized QconCATs, MEERCAT can co-synthesize up to 150 QconCATs in a single reaction. In a quantitative proteomics study using the MEERCAT approach, it was possible for one experimenter to establish a standard peptide library targeting ~1,500 yeast proteins in two days.