title: Statistical model to analyze quantitative proteomics data obtained by 18O/16O labeling and linear ion trap mass spectrometry: application to the study of vascular endothelial growth factor-induced angiogenesis in endothelial cells.
abstract: Statistical models for the analysis of protein expression changes by stable isotope labeling are still poorly developed, particularly for data obtained by 16O/18O labeling. Besides large scale test experiments to validate the null hypothesis are lacking. Although the study of mechanisms underlying biological actions promoted by vascular endothelial growth factor (VEGF) on endothelial cells is of considerable interest, quantitative proteomics studies on this subject are scarce and have been performed after exposing cells to the factor for long periods of time. In this work we present the largest quantitative proteomics study to date on the short term effects of VEGF on human umbilical vein endothelial cells by 18O/16O labeling. Current statistical models based on normality and variance homogeneity were found unsuitable to describe the null hypothesis in a large scale test experiment performed on these cells, producing false expression changes. A random effects model was developed including four different sources of variance at the spectrum-fitting, scan, peptide, and protein levels. With the new model the number of outliers at scan and peptide levels was negligible in three large scale experiments, and only one false protein expression change was observed in the test experiment among more than 1000 proteins. The new model allowed the detection of significant protein expression changes upon VEGF stimulation for 4 and 8 h. The consistency of the changes observed at 4 h was confirmed by a replica at a smaller scale and further validated by Western blot analysis of some proteins. Most of the observed changes have not been described previously and are consistent with a pattern of protein expression that dynamically changes over time following the evolution of the angiogenic response. With this statistical model the 18O labeling approach emerges as a very promising and robust alternative to perform quantitative proteomics studies at a depth of several thousand proteins.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, E-28049 Madrid, Spain and ||Centro Nacional de Investigaciones Cardiovasculares, E-28029 Madrid, Spain.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Jorge
authors.forename: Pedro
authors.initials: P
authors.lastname: Navarro
authors.forename: Pablo
authors.initials: P
authors.lastname: Martínez-Acedo
authors.forename: Estefanía
authors.initials: E
authors.lastname: Núñez
authors.forename: Horacio
authors.initials: H
authors.lastname: Serrano
authors.forename: Arántzazu
authors.initials: A
authors.lastname: Alfranca
authors.forename: Juan Miguel
authors.initials: JM
authors.lastname: Redondo
authors.forename: Jesús
authors.initials: J
authors.lastname: Vázquez
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008715:Methionine / Q000378:metabolism; D015233:Models, Statistical; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010084:Oxidation-Reduction / Q000187:drug effects; D010103:Oxygen Isotopes; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D040901:Proteomics; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
chemical_list: D010103:Oxygen Isotopes; D010455:Peptides; D042461:Vascular Endothelial Growth Factor A; D008715:Methionine