Abstract
High-sensitivity mass spectrometry approaches using selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems. Both high-sensitivity detection of low-abundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards. Currently, there are various ways for preparing standards, including chemical peptide synthesis, cellular protein expression, and cell-free protein or peptide synthesis. Cell-free protein synthesis (CFPS) or in vitro translation (IVT) systems in particular provide high-throughput and low-cost preparation methods, and various cell types and reconstituted forms are now commercially available. Herein, we review the use of such systems for precise and reliable protein quantification.
Highlights
For selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) methods that are frequently employed in targeted proteomics experiments, selection of both precursor and fragment ions by a triple quadrupole mass spectrometer is necessary to limit the number of peptides observed for each target, which enables highly sensitive analysis of target peptides in samples
In recent years, targeted quantitative proteomics using internal standards synthesized by cell-free protein synthesis (CFPS) or in vitro translation (IVT) systems have gradually attracted attention
This review focuses on the cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics studies
Summary
In recent years, targeted quantitative proteomics using internal standards synthesized by cell-free protein synthesis (CFPS) or in vitro translation (IVT) systems have gradually attracted attention. This review focuses on the cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics studies. Stable isotope labeling can be introduced nonspecifically to intact cell proteins, enabling various peptides to be synthesized simultaneously, which is more effective for global proteomics analysis.
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