Overview
Purpose: Examine the effect of pressure cycling technology (PCT) on proteolysis cleavage efficiency, reaction time, and protein sequence coverage for LC-MS/MS analyses.
Methods: Protein samples were digested with proteases under pressure cycling conditions and under standard digestion conditions. The resulting peptides were analyzed using LC-MS/MS with an ion trap mass spectrometer (MS) equipped with electron transfer dissociation (ETD) or an ion trap-Orbitrap hybrid MS.
Results: PCT digestion protocols using trypsin, chymotrypsin, LysC and GluC provided comparable sequence coverage to standard overnight digests. PCT digestion of complex mixtures of cytosolic proteins produced similar results. PCT improved the digestion of post-nuclear membrane proteins.