Determination of variation parameters as a crucial step in designing TMT-based clinical proteomics experiments

PloS One
Evelyne MaesInge Mertens

Abstract

In quantitative shotgun proteomic analyses by liquid chromatography and mass spectrometry, a rigid study design is necessary in order to obtain statistically relevant results. Hypothesis testing, sample size calculation and power estimation are fundamental concepts that require consideration upon designing an experiment. For this reason, the reproducibility and variability of the proteomic platform needs to be assessed. In this study, we evaluate the technical (sample preparation), labeling (isobaric labels), and total (biological + technical + labeling + experimental) variability and reproducibility of a workflow that employs a shotgun LC-MS/MS approach in combination with TMT peptide labeling for the quantification of peripheral blood mononuclear cell (PBMC) proteome. We illustrate that the variability induced by TMT labeling is small when compared to the technical variation. The latter is also responsible for a substantial part of the total variation. Prior knowledge about the experimental variability allows for a correct design, a prerequisite for the detection of biologically significant disease-specific differential proteins in clinical proteomics experiments.

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Citations

Feb 18, 2016·Obesity Reviews : an Official Journal of the International Association for the Study of Obesity·L MertensI De Bourdeaudhuij
Aug 19, 2015·Critical Reviews in Oncology/hematology·Evelyne MaesGeert Baggerman
Jun 16, 2016·Molecular & Cellular Proteomics : MCP·Evelyne MaesDirk Valkenborg
Sep 22, 2018·Oncotarget·Rebeca KawaharaGiuseppe Palmisano
Feb 19, 2021·Lipids in Health and Disease·Lishann M IngramBrian S Cummings
Mar 5, 2021·Journal of Proteome Research·Neil A McCrackenAmber L Mosley

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Methods Mentioned

BETA
proteomic profiling
protein Assay
reversed phase chromatography

Software Mentioned

Sequest
MASCOT
Proteome discoverer

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