Pulse Proteolysis and Precipitation for Target Identification

In
recent years, phenotypic screening has assumed a leading role
in drug discovery efforts. However, development of new drugs from
bioactive compounds obtained in screening campaigns requires identification
of the cellular targets responsible for their biological activities.
A new energetics-based method for target identification is presented:
pulse proteolysis and precipitation for target identification (PePTID).
In this method, proteins incubated with or without a ligand and submitted
to a brief proteolytic pulse are directly analyzed and compared using
a label-free semiquantitative mass spectrometry strategy, dispensing
the SDS-PAGE readout and greatly improving the throughput. As a proof-of-concept,
we applied the PePTID method to identify ATP-binding proteins in Mycobacterium smegmatis, a model system for Mycobacterium
tuberculosis, the etiological agent of tuberculosis.