title: Rapid biosynthesis of stable isotope-labeled peptides from a reconstituted in vitro translation system for targeted proteomics.
abstract: 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.
authors.affiliation: CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, PR China; BGI-Shenzhen, Shenzhen, PR China; Sino-Danish Center/Sino-Danish College, University of Chinese Academy of Sciences, Beijing, PR China.
authors.forename: Feng
authors.initials: F
authors.lastname: Xian
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland, USA. Electronic address: sli@umd.edu.
authors.forename: Shuwei
authors.initials: S
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, PR China; BGI-Shenzhen, Shenzhen, PR China; Sino-Danish Center/Sino-Danish College, University of Chinese Academy of Sciences, Beijing, PR China. Electronic address: siqiliu@genomics.cn.
authors.forename: Siqi
authors.initials: S
authors.lastname: Liu
mesh_terms: D066298:In Vitro Techniques; D007553:Isotope Labeling; D010455:Peptides / Q000008:administration & dosage*; D014176:Protein Biosynthesis; D040901:Proteomics
chemical_list: D010455:Peptides
keywords: Isotope-labeled reference peptides; Mass spectrometry; Protein quantitation; Reconstituted in vitro translation system; Targeted proteomics