Fast parallel proteolysis (FASTpp) is a method to determine

.the thermostabilityof proteins that does not need previous purification and labelling

History and background[edit]

Proteolysis is widely used in biochemistry and cell biology to probe protein structure.
[2][3]

In "limited trypsin proteolysis", low amounts of protease digest both foldedand

unfolded protein but at largely different rates: unstructured proteins are cut more
rapidly, while structured proteins are cut at a slower rate (sometimes by orders of
magnitude). Recently, several other assays of protein stability based on proteolysis
have been proposed, exploiting other proteases with high specificity for cleaving
unfolded proteins. These include Pulse Proteolysis,[4] Proteolytic Scanning
Calorimetry [5] and FASTpp.

How it works[edit]
FASTpp measures the quantity of protein that resists digestion under various
conditions. To this end, a thermostable protease is used, which cleaves specifically at
exposed hydrophobic residues. The FASTpp assay combines the thermal unfolding,
specificity of a thermostable protease for the unfolded fraction with the separation
power of SDS-PAGE.[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
cytosolic proteome. Applications range from biotechnology to study of point
mutationsand ligand binding assays.

Applications[edit]
FASTpp has been used to probe:[1]

Lysate effect on protein stability

Coupled folding and binding [7]

Ligand effects on fraction folded & stability [8]

Effects of mutations on fraction folded & stability (e.g. point

mutation/missense mutationss[8][9])

Kinetic protein stability [10]

Principles[edit]

A protein mixture is aliquoted into several tubes, which are exposed in parallel to
different temperatures and a thermostable protease (see figure). Automated
temperature control is achieved in a thermal gradient cycler (commonly used
for PCRs). Reaction products can be separated by SDS-PAGE or western blot.[6] The
protease thermolysin can be fully inactivated by EDTA. This feature
of thermolysin makes FASTpp compatible with subsequent trypsin digestion e.g.
for mass spectrometry.[11][12]