title: Label-free quantification and shotgun analysis of complex proteomes by one-dimensional SDS-PAGE/NanoLC-MS: evaluation for the large scale analysis of inflammatory human endothelial cells.
abstract: To perform differential studies of complex protein mixtures, strategies for reproducible and accurate quantification are needed. Here, we evaluated a quantitative proteomic workflow based on nanoLC-MS/MS analysis on an LTQ-Orbitrap-VELOS mass spectrometer and label-free quantification using the MFPaQ software. In such label-free quantitative studies, a compromise has to be found between two requirements: repeatability of sample processing and MS measurements, allowing an accurate quantification, and high proteomic coverage of the sample, allowing quantification of minor species. The latter is generally achieved through sample fractionation, which may induce experimental bias during the label-free comparison of samples processed, and analyzed independently. In this work, we wanted to evaluate the performances of MS intensity-based label-free quantification when a complex protein sample is fractionated by one-dimensional SDS-PAGE. We first tested the efficiency of the analysis without protein fractionation and could achieve quite good quantitative repeatability in single-run analysis (median coefficient of variation of 5%, 99% proteins with coefficient of variation <48%). We show that sample fractionation by one-dimensional SDS-PAGE is associated with a moderate decrease of quantitative measurement repeatability while largely improving the depth of proteomic coverage. We then applied the method for a large scale proteomic study of the human endothelial cell response to inflammatory cytokines, such as TNFα, interferon γ, and IL1β, which allowed us to finely decipher at the proteomic level the biological pathways involved in endothelial cell response to proinflammatory cytokines.
authors.affiliation: Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France.
authors.forename: Violette
authors.initials: V
authors.lastname: Gautier
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Mouton-Barbosa
authors.forename: David
authors.initials: D
authors.lastname: Bouyssié
authors.forename: Nicolas
authors.initials: N
authors.lastname: Delcourt
authors.forename: Mathilde
authors.initials: M
authors.lastname: Beau
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Girard
authors.forename: Corinne
authors.initials: C
authors.lastname: Cayrol
authors.forename: Odile
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.forename: Bernard
authors.initials: B
authors.lastname: Monsarrat
authors.forename: Anne
authors.initials: A
authors.lastname: Gonzalez de Peredo
mesh_terms: D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015870:Gene Expression / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D018836:Inflammation Mediators; D053583:Interleukin-1beta; D011506:Proteins; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma