Stable isotope-labeled peptides are routinely used as internal standards (a.k.a. reference peptides) for absolute quantitation of proteins in targeted proteomics. These peptides can either be synthesized chemically on solid supports or expressed biologically by concatenating multiple peptides together to a large protein. Neither method, however, has required versatility, convenience, and economy for making a large number of reference peptides. Here, we describe the biosynthesis of stable isotope-labeled peptides from a reconstituted Escherichia coli in vitro translation system. We provide a detailed protocol on how to express these peptides with high purity and how to determine their concentrations with easiness. Our strategy offers a general, fast, and scalable approach for the easy preparation of labeled reference peptides, which will have broad application in both basic research and translational medicine.
Keywords: Isotope-labeled reference peptides; Mass spectrometry; Protein quantitation; Reconstituted in vitro translation system; Targeted proteomics.
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