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Mol Cell Proteomics. 2016 Aug;15(8):2819-28. doi: 10.1074/mcp.O115.056507. Epub 2016 May 27.

Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

Author information

1
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;
2
§BGI-Shenzhen, Shenzhen, 518083, China;
3
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;
4
From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China;
5
**Beijing Protein Innovation, Beijing, 101318, China.
6
‖Institute for Bioscience and Biotechnology Research, University of Maryland College Park, Rockville, Maryland 20850; siqiliu@genomics.cn sli@umd.edu.
7
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.

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.

PMID:
27234506
PMCID:
PMC4974354
DOI:
10.1074/mcp.O115.056507
[Indexed for MEDLINE]
Free PMC Article

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