In 2012 selective reaction monitoring (SRM) was Nature methods (link is external) method of the year. A new paper (link is external) from VIB and Ghent University builds on this technique to be able to easily and accurately quantify cellular proteins in any organism and claims that this will spell the end to the traditional biochemical assays like ELISA and western blots.
Professor Sven Eyckerman and colleagues have developed a set of universal protein tags that warrant protein quantification via targeted proteomics techniques – named Proteotypic peptides for Quantification by SRM (PQS).
Getting an accurate view on the cellular concentration of a protein is a challenging task. Affinity reagent-based approaches like ELISA and western blotting are sensitive and convenient, but inadequate antibodies and epitope masking can interfere with detection. Instead quantification by SRM doesn’t rely on immunoaffinity reagents, but uses targeted mass spectrometry to monitor the expression levels of a protein. Despite the clear use for targeted proteomics, assay development remains challenging as multiple peptides need to be monitored for each protein studied to account for interference from potential PTMs. Also, the selected peptides have to be unique for the protein given the background proteome in which the protein of interest is being analysed.
Unique peptide sequences with optimal mass spectrometry characteristics were identified from the hyperthermophile Pyrococcus furiosus. To make sure any identified peptides would be universally applicable the team purposefully looked for peptides unique amongst all eukaryotes and E.coli strains. Two peptides were found that are suitable for direct sensitive detection and quantification in complex lysates. Using these peptides in SRM removes the need to set-up an individual SRM assay for each protein being studied, which was labour intensive and often had a low success rate.
To introduce the quantifiable tags into the genome of human cells, the scientists put new recruits of the genome engineering toolbox into action. Using the CRISP/Cas9 system they tagged endogenous proteins in mammalian cell lines and were able to provide a universal quantitative read-out system for the tagged proteins and this technology can be used either directly or following purification of the protein within a protein complex.
With their new PQS for SRM the Belgian team have almost removed the assay development time for SRM analysis, though the CRISPR element of the protocol remains to be optimised. Mass spectrometry-based methods have typically been perceived to hold the advantage over antibody techniques in terms of development times, due to the long development time of antibody reagents themselves, but the rapid development time of Affimer reagents of just weeks rather than months brings these factors into parallel.
Conversely, affinity reagents have been deemed to have the upper hand in terms of sensitivity of detection of low abundance proteins, yet targeted proteomic approaches may now be able to correct for part of the sensitivity issues from mass spectrometry. What’s more issues of detection specificity associated with antibodies can be overcome with Affimer reagents, as negative selection for homologous proteins can be easily incorporated into our protein screens.
Perhaps the playing field between these different approaches is levelling. With applications in research and therapeutics affinity reagent development remains one of the fastest growing sectors in the pharmaceutical industry. Whether the simpler but equally effective immunoaffinity protein quantification techniques of ELISA and western blotting will be superseded by more complex CRISPR and SRN protocols remains to be seen.