Our vision and the technology behind the Protein Turnover Atlas

A crucial yet challenging part of drug discovery and development is fully understanding a compound of interest’s potency, selectivity, target space, and pharmacokinetics/pharmacodynamics (PK/PD).

The Protein Turnover Atlas (PTA) provides essential information to support those efforts, as the effect of a drug is not only influenced by the presence of the compound but also depends on the availability of target proteins of interest. Having knowledge about protein half-life is therefore essential and can guide the selection of potential targets and modalities. It provides a key measure to evaluate the relevance of off-targets, and it represents a crucial parameter in PK/PD.

Proteomics is a key technology that can not only measure the abundance of thousands of proteins in a single sample but also enables protein half-lives to be determined on a global scale.

At OmicScouts, we developed a workflow called TurnoverScout. It is an in vitro time-course assay applicable to any type of cell line. The application of stable isotope labelling of amino acids in cell culture (pSILAC) allows us to track time-dependent processes like protein degradation.

Initially, cells are allowed to proliferate without any constraints. At time point zero, we switch the cell medium from so-called light medium to heavy medium. The two media differ in the isotopic state of two amino acids – arginine and lysine. While Lys0 and Arg0 are present in the light medium, the heavy medium contains their heavier counterparts, Lys8 and Arg10. This difference in mass can be detected and quantified with mass spectrometry.

After the medium switch, only heavy amino acids are available for protein synthesis (neglecting recycling). As a consequence, the existing protein pool is gradually replaced with newly synthesized heavy-labelled versions of the same proteins until they have been fully replaced. The dynamics of light-protein disappearance enable us to determine the individual degradation rate for each protein.

In our classical setup, cells are labeled for up to 7 days, harvested, and prepared via automated workflow. Cells are digested with trypsin to generate peptides that contain at least one lysine or arginine (either light or heavy versions) and quantified via mass spectrometry.

Search engines like MaxQuant map these peptides to proteins based on a library and allow us to determine the abundance of both states of a protein by considering the defined mass shifts.

The workflow is complemented by applying a simple mathematical model which describes the exponential dynamics of the light and heavy protein pools as the interplay between protein degradation and synthesis for each protein. By scaling and fitting the data to that model, we can estimate the clearance/degradation rate under the steady-state assumption and, subsequently, the protein half-life.