title: Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.
abstract: Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible.
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; §BGI-Shenzhen, Shenzhen, 518083, China; ¶Sino-Danish Center for Education and Research, University of the Chinese Academy of Sciences, Beijing, 100049, China;
authors.forename: Feng
authors.initials: F
authors.lastname: Xian
authors.affiliation: §BGI-Shenzhen, Shenzhen, 518083, China;
authors.forename: Jin
authors.initials: J
authors.lastname: Zi
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; §BGI-Shenzhen, Shenzhen, 518083, China;
authors.forename: Quanhui
authors.initials: Q
authors.lastname: Wang
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China;
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Lou
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China;
authors.forename: Haidan
authors.initials: H
authors.lastname: Sun
authors.affiliation: §BGI-Shenzhen, Shenzhen, 518083, China;
authors.forename: Liang
authors.initials: L
authors.lastname: Lin
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; §BGI-Shenzhen, Shenzhen, 518083, China;
authors.forename: Guixue
authors.initials: G
authors.lastname: Hou
authors.affiliation: §BGI-Shenzhen, Shenzhen, 518083, China;
authors.forename: Weiqiao
authors.initials: W
authors.lastname: Rao
authors.affiliation: **Beijing Protein Innovation, Beijing, 101318, China.
authors.forename: Changcheng
authors.initials: C
authors.lastname: Yin
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China;
authors.forename: Lin
authors.initials: L
authors.lastname: Wu
authors.affiliation: ‖Institute for Bioscience and Biotechnology Research, University of Maryland College Park, Rockville, Maryland 20850; siqiliu@genomics.cn sli@umd.edu.
authors.forename: Shuwei
authors.initials: S
authors.lastname: Li
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; §BGI-Shenzhen, Shenzhen, 518083, China; ¶Sino-Danish Center for Education and Research, University of the Chinese Academy of Sciences, Beijing, 100049, China; siqiliu@genomics.cn sli@umd.edu.
authors.forename: Siqi
authors.initials: S
authors.lastname: Liu
mesh_terms: D000818:Animals; D002474:Cell-Free System; D002853:Chromatography, Liquid / Q000379:methods; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*
chemical_list: D010455:Peptides