Fast parallel proteolysis

[6] Due to this combination, FASTpp can detect changes in the fraction folded over a large physico-chemical range of conditions including temperatures up to 85 °C, pH 6–9, presence or absence of the whole proteome.

FASTpp has been used to probe:[1] First, a cell lysate is generated by glass beads beating, pressure homogenisation or chemical or physical lysis methods that do not denature the protein(s) of interest.

(Optionally for targeted analysis) a protein of interest is purified out of this lysate by affinity methods based on intrinsically disordered tags [12] or other suitable purification strategies, often involving several orthogonal chromatographic steps.

All aliquots are exposed in parallel in a thermal gradient PCR cycler to different maximal temperatures in presence of the thermostable protease thermolysin (see figure).

This feature of thermolysin makes FASTpp compatible with subsequent trypsin digestion e.g. for mass spectrometry.

FASTpp (Fast parallel proteolysis). A protein mixture is aliquoted into several tubes, which are exposed in parallel to different temperatures and a thermostable protease. The remaining protein can be resolved on SDS-PAGE .