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Updated: Mar 20, 2026
Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
Published on: August 17, 2015
Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with
Feng Xian1, Jin Zi2, Quanhui Wang3
1From 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;
Generating isotope-labeled peptides for quantitative targeted proteomics is challenging. This study presents an in vitro expression system for synthesizing these peptides, enabling accurate quantification and cost-effective large-scale applications in proteomics research.
Area of Science:
- Proteomics
- Biochemistry
- Mass Spectrometry
Background:
- Targeted proteomics using mass spectrometry and multiple reaction monitoring (MRM) is crucial for peptide detection and quantification.
- Absolute quantification of peptides relies on isotope-labeled internal standards, but their generation is a significant challenge for large-scale quantitative proteomics.
- Existing methods for producing isotope-labeled peptides are often costly and complex, hindering widespread adoption.
Purpose of the Study:
- To develop a novel and efficient method for synthesizing isotope-labeled peptides using an in vitro expression system.
- To create a system for enriching and quantifying these synthesized peptides using fused quantitative tags.
- To demonstrate the utility of these recombinant peptides as internal standards for absolute quantification in complex biological samples.
Main Methods:
- Utilized an Escherichia coli cell-free protein expression system (protein synthesis using recombinant elements) to synthesize isotope-labeled peptides fused with a Strep-tag.
- Optimized expression conditions for efficient synthesis of labeled peptides.
- Employed Strep-Tactin affinity enrichment for peptide purification.
- Developed a method for complete tryptic digestion to release the Strep-tag for quantification.
- Applied the synthesized isotope-labeled peptides as internal standards for quantitative mass spectrometry.
Main Results:
- Successfully synthesized isotope-labeled peptides fused with Strep-tag using an in vitro cell-free system.
- Achieved acceptable yields of labeled peptides after affinity enrichment, suitable for large-scale quantification.
- Demonstrated that the recombinant peptides function effectively as internal standards in multiple reaction monitoring assays.
- Validated the method by accurately quantifying glutathione S-transferase (GST) in mouse serum samples, showing measurable and reproducible results.
Conclusions:
- The proposed in vitro expression system offers an economical and efficient approach for generating isotope-labeled peptides for targeted proteomics.
- This method overcomes the challenges associated with traditional isotope-labeled peptide synthesis, facilitating large-scale quantitative proteomics.
- The recombinant peptides are versatile internal standards, enabling accurate and reproducible absolute quantification in complex biological matrices.
