Comparative Analysis of Mass-Spectrometry-Based Proteomic Methods for Protein Target Discovery Using a One-Pot Approach

Recently, several mass-spectrometry- and protein-denaturation-based
proteomic methods have been developed to facilitate protein target
discovery efforts in drug mode-of-action studies. These methods, which
include the stability of proteins from rates of oxidation (SPROX),
pulse proteolysis (PP), chemical denaturation and protein precipitation
(CPP), and thermal proteome profiling (TPP) techniques, have been
used in an increasing number of applications in recent years. However,
while the advantages and disadvantages to using these different techniques
have been reviewed, the analytical characteristics of these methods
have not been directly compared. Reported here is such a direct comparison
using the well-studied immunosuppressive drug, cyclosporine A (CsA),
and the proteins in a yeast cell lysate. Also described is a one-pot
strategy that can be utilized with each technique to streamline data
acquisition and analysis. We find that there are benefits to utilizing
all four strategies for protein target discovery including increased
proteomic coverage and reduced false positive rates that approach
0%. Moreover, the one-pot strategy described here makes such an experiment
feasible, because of the 10-fold reduction in reagent costs and instrument
time it affords.