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

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Cabrera, A; Wiebelhaus, N; Quan, B; Ma, R; Meng, H; Fitzgerald, MC

Published Date

  • February 2020

Published In

Volume / Issue

  • 31 / 2

Start / End Page

  • 217 - 226

PubMed ID

  • 32031398

Pubmed Central ID

  • PMC7441748

Electronic International Standard Serial Number (EISSN)

  • 1879-1123

International Standard Serial Number (ISSN)

  • 1044-0305

Digital Object Identifier (DOI)

  • 10.1021/jasms.9b00041


  • eng