Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides.

Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis. SIGNIFICANCE: The developed method can be applied for the preparation of internal standard peptides without limiting the number of peptides to be synthesized, which may result in more practical screening of quantitatively reliable peptides, one of the fundamental steps in the reliable absolute quantification using MS. Furthermore, the method is highly versatile for proteome analysis of any organisms or species without any cDNA or SIL peptide libraries. The quantification can be finished in a few days including design and preparation of appropriate SIL peptides using small-scale batch cell-free reactions, which has a potential to be a part of the standard methodology in a field of quantitative proteomics.