title: Assessing reproducibility of a protein dynamics study using in vivo labeling and liquid chromatography tandem mass spectrometry.
abstract: Measuring dynamics of proteins abundance in cells in response to stimuli such as growth factors or drugs requires analysis of more than one time point. Proteomic approaches have traditionally been used to measure only one state at a time because quantitation is difficult, especially when mass spectrometry is used as a readout. Isotopically labeled reagents have recently been introduced that allow comparison of two or three different states by mass spectrometry. Here, we evaluate the reproducibility of an experiment that measures three states simultaneously through stable isotope labeling of cells with amino acids in cell culture (SILAC) using light, medium, and heavy versions of amino acids. The major goal of this study was to assess the reproducibility of such experiments in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS). Our results show that it is possible to obtain reproducible quantitative data to study protein dynamics based on our analysis of more than 220 peptide sets derived from 20 proteins from 3 different LC-MS/MS runs.
authors.affiliation: McKusick-Nathans Institute for Genetic Medicine and the Department of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland 21205, USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.forename: Giovanni
authors.initials: G
authors.lastname: Parmigiani
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
mesh_terms: D001120:Arginine / Q000378:metabolism; D002853:Chromatography, Liquid; D006367:HeLa Cells; D006801:Humans; D009218:Myosins / Q000378:metabolism; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D020652:Peptide Elongation Factor 2; D011506:Proteins; D001120:Arginine; D009218:Myosins