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

Abstract

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 immuno-suppressive 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.

Chemoproteomic-enabled characterization of small GTPase Rab1a as a target of anN-arylbenzdiimidazole ligand’s rescue of Parkinson’s-associated cell toxicity

“…To employ these techniques for identification of NAB2 protein targets with high confidence and in an efficient manner, a highly multiplexed “one-pot” method was used. This method, which was recently validated by Cabrera and co-workers, 30 uses cellular lysate in the presence or absence of small molecule treatment ( Figure 2C ). The lysate samples are processed according to standard TPP or SPROX workflows, where the samples (five biological replicates of each NAB2- or vehicle-treated control) are exposed to a denaturation gradient (temperature for TPP and urea for SPROX) followed by chemical oxidation (SPROX) or ultra-centrifugation (TPP).…”

“…Though the one-pot approach precludes generation of full denaturation curves for proteins identified, it enables high confidence identification of hit proteins through a high number of biological replicates per method, which minimizes not only the experimental cost but also the false positive rate. 30 Requiring hit proteins to appear in multiple orthogonal methods (e.g., TPP and SPROX) also has been shown in model studies to result in false positive rates close to 0. SPROX measures the stability of proteins as a function of the protein susceptibility to chemical oxidation over a chemical denaturation gradient.…”

“…(C) Cabrera and co-workers recently reported a one-pot workflow for combinatorial analysis of two orthogonal chemoproteomic experiments in a single sample for LC-MS/MS analysis. 30 This workflow was employed with TPP/SPROX to identify NAB2 targets.…”

“…To this end, we employed a chemoproteomic strategy in which a multiplexed, one-pot approach enabled parallel TPP and SPROX identification of proteins that exhibit NAB2-dependent shifts in stability. 30 Through this analysis, we identified the small GTPase, Rab1a, as a hit in both TPP and SPROX analyses, providing Rab1a as a putative, previously unrecognized target of NAB2. The identification of Rab1a as a target in the rescue of α-synuclein toxicity is especially interesting as Rab1a regulates trafficking from the ER to Golgi, a process disrupted by α-synuclein toxicity but rescued by NAB2 in phenotypic analyses.…”

“…In SPROX, methionine residues are specifically oxidized in the presence of H2O2, and shifts in protein stability are measured as a function of changes in the midpoint of methionine oxidation curves. (C) Cabrera and co-workers recently reported a one-pot workflow for combinatorial analysis of two orthogonal chemoproteomic experiments in a single sample for LC-MS/MS analysis 30. This workflow was employed with TPP/SPROX to identify NAB2 targets.…”