Targeted Quantification

Targeted strategies have emerged ten years ago and are best suited when precise, ideally absolute, quantification of finite sets of proteins are required with highest sensitivity. Targeted strategies are widely applied in the biomarker field as potential biomarker candidates identified during discovery experiments need to be verified and validated on large sample cohorts, sometimes even in a clinical environment.

The ProFI teams have expertise in targeted proteomics for the development of robust targeted assays using Selected/Multiple Reaction Monitoring (SRM/MRM) on triple-quadrupole-type instruments or high resolution accurate mass targeted monitoring modes such as Parallel Reaction Monitoring (PRM) or DIA (Data Independent Acquisition) on high resolution instruments. In that context our objectives are to provide precise and absolute quantification of finite sets of proteins of interest, to develop a robust targeted quantification assay and to transfer quantification assays to the clinics for biomarker monitoring. We develop targeted assays on complex protein mixtures (whole cell lysates, blood, plasma, urine, CSF etc.), enzymatically digested (e.g. trypsin) and eventually fractionated at protein and/or peptide levels. The resulting peptide mixtures are analyzed on liquid chromatography systems (nano- or micro-scale) coupled to dedicated mass spectrometers run in Selected/Multiple Reaction Monitoring (SRM/MRM) or high resolution full scan MS/MS (HR-MRM, HD-MRM, PRM or DIA) modes. According to the project, we design optimized analytical workflows including the following steps:

  • Choice of proteins and peptides to be targeted. We use all existing resources to refine the choice of tracer peptides, including prior shotgun data screening (in-house acquired or available on ProteomeExchange), public and dedicated repositories (such as SRMAtlas, PASSEL), software for prediction
  • Sample preparation. We can implement various protein extraction methods, as for discovery experiments. Of note is that for a robust quantification, sample preparation should be minimal as each step introduces additional variability.
  • Design and synthesis of heavy labelled standards (e.g. AQUA peptides, PSAQ standards).
  • LC-MS-MS analysis. We use state-of-the art LC-MS/MS couplings, based on powerful chromatographic systems and last generation mass spectrometers that are benchmarked and quality controlled to ensure a sustained level of performance and with optimum chromatographic gradients, chromatographic scales (nano or micro) and MS modes.