title: Determination of variation parameters as a crucial step in designing TMT-based clinical proteomics experiments.
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.
authors.affiliation: Flemish Institute for Technological Research (VITO), Mol, Belgium; CFP-CeProMa, University of Antwerp, Antwerp, Belgium; KU Leuven, Research Group of Functional Genomics and Proteomics, Leuven, Belgium.
authors.forename: Evelyne
authors.initials: E
authors.lastname: Maes
authors.affiliation: Flemish Institute for Technological Research (VITO), Mol, Belgium; CFP-CeProMa, University of Antwerp, Antwerp, Belgium; Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Diepenbeek, Belgium.
authors.forename: Dirk
authors.initials: D
authors.lastname: Valkenborg
authors.affiliation: Flemish Institute for Technological Research (VITO), Mol, Belgium; CFP-CeProMa, University of Antwerp, Antwerp, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: Baggerman
authors.affiliation: Flemish Institute for Technological Research (VITO), Mol, Belgium; CFP-CeProMa, University of Antwerp, Antwerp, Belgium.
authors.forename: Hanny
authors.initials: H
authors.lastname: Willems
authors.affiliation: KU Leuven, Research Group of Functional Genomics and Proteomics, Leuven, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Landuyt
authors.affiliation: KU Leuven, Research Group of Functional Genomics and Proteomics, Leuven, Belgium.
authors.forename: Liliane
authors.initials: L
authors.lastname: Schoofs
authors.affiliation: Flemish Institute for Technological Research (VITO), Mol, Belgium; CFP-CeProMa, University of Antwerp, Antwerp, Belgium.
authors.forename: Inge
authors.initials: I
authors.lastname: Mertens
mesh_terms: D016000:Cluster Analysis; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000378:metabolism; D010865:Pilot Projects; D020543:Proteome; D040901:Proteomics / Q000379:methods* / Q000592:standards*; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D020543:Proteome