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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 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.

SUBMITTER: Cabrera A

PROVIDER: S-EPMC7441748 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Cabrera Aurora A Wiebelhaus Nancy N Quan Baiyi B Ma Renze R Meng He H Fitzgerald Michael C MC

Journal of the American Society for Mass Spectrometry 20191122 2

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 advantag ...[more]

PMID: 32031398

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Project description:BackgroundSample preparation is a critical process for proteomic studies. Many efficient and reproducible sample preparation methods have been developed for mass spectrometry-based proteomic analysis of human and animal tissues or cells, but no attempt has been made to evaluate these protocols for plants. We here present an LC-MS/MS-based proteomics study of barley leaf aimed at optimization of methods to achieve efficient and unbiased trypsin digestion of proteins prior to LC-MS/MS based sequencing and quantification of peptides. We evaluated two spin filter-aided sample preparation protocols using either sodium dodecyl-sulphate or sodium deoxycholate (SDC), and three in-solution digestion (ISD) protocols using SDC or trichloroacetic acid/acetone precipitation.ResultsThe proteomics workflow identified and quantified up to 1800 barley proteins based on sequencing of up to 6900 peptides per sample. The two spin filter-based protocols provided a 12-38% higher efficiency than the ISD protocols, including more proteins of low abundance. Among the ISD protocols, a simple one-step reduction and S-alkylation method (OP-ISD) was the most efficient for barley leaf sample preparation; it identified and quantified 1500 proteins and displayed higher peptide-to-protein inference ratio and higher average amino acid sequence coverage of proteins. The two spin filter-aided sample preparation protocols are compatible with TMT labelling for quantitative proteomics studies. They exhibited complementary performance as about 30% of the proteins were identified by either one or the other protocol, but also demonstrated a positive bias for membrane proteins when using SDC as detergent.ConclusionsWe provide detailed protocols for efficient plant protein sample preparation for LC-MS/MS-based proteomics studies. Spin filter-based protocols are the most efficient for the preparation of leaf samples for MS-based proteomics. However, a simple protocol provides comparable results although with different peptide digestion profile.

Dataset Information

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

Publications

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

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