NEW YORK (GenomeWeb) — A team led by researchers from the Beijing Institute of Genomics and BGI-Shenzhen has devised a cell-free protein expression system to produce isotope-labeled peptides for use in targeted peptide quantitation assays.
Detailed in a study published last month in Molecular & Cellular Proteomics, the approach uses an Escherichia coli cell-free protein expression system and could enable easier and larger-scale production of labeled peptides, the authors wrote.
In multiple-reaction monitoring experiments, researchers use triple quadrupole mass specs to quantify specific peptides which can then be used as proxies for the levels of larger proteins. Typically, stable isotope-labeled versions of the peptides being measures are spiked into the experiment and used as internal standards, increasing the precision of the experiment.
This means that targeted MRM assays are dependent on the production of the necessary peptides standards, which have traditionally been produced via solid-phase peptide synthesis. As the authors of this new study noted, however, this approach is relatively tedious and expensive. Additionally, some peptide sequences are difficult to synthesize and purify.
Due to these limitations, researchers have begun exploring biological approaches using protein expression systems such as the QconCAT platform, which uses oligonucleotides coding for a protein containing the desired peptides inserted into a concatenated gene that is then expressed in E. coli grown in a labeled medium. The expressed protein is then purified and digested into the separate peptides.
This approach also has limitations, however, including the fact that some genes cannot be expressed and that some of the protein products may be digested by non-specific proteases in the E. coli.
Hoping to improve upon this approach, the authors adopted the PURE cell-free system consisting of E. coli. translational machinery. The approach, they noted has certain advantages, including the fact that the system lacks proteases and enzymes for post-translational modifications, eliminating the concern that peptide standards might be degraded or modified. It is also faster than the QconCAT approach, they wrote, taking from one to two days from the initial design to the final peptide quantitation.
One limitation of the approach is yield, which could prove to be an issue for experiments where large amounts of a peptide standard are required, the team noted. However, the process can be performed in a 96-well plate format, which could help with generating standards for larger scale experiments.
In experiments testing reagents developed by the method for measuring glutathione S-transferase levels in serum, the researchers found that four GST standards generated using the PURE system proved to be effective internals standards for GST quantification.