title: Two Birds with One Stone: Parallel Quantification of Proteome and Phosphoproteome Using iTRAQ.
abstract: Altered and abnormal levels of proteins and their phosphorylation states are associated with many disorders. Detection and quantification of such perturbations may provide a better understanding of pathological conditions and help finding candidates for treatment or biomarkers. Over the years, isobaric mass tags for relative quantification of proteins and protein phosphorylation by mass spectrometry have become increasingly popular. One of the most commonly used isobaric chemical tags is iTRAQ (isobaric tag for relative and absolute quantitation). In a typical iTRAQ-8plex experiment, a multiplexed sample amounts for up to 800 μg of peptides. Using state-of-the-art LC-MS approaches, only a fraction (~5 %) of such a sample is required to generate comprehensive quantitative data on the global proteome level, so that the bulk of the sample can be simultaneously used for quantitative phosphoproteomics. Here, we provide a simple and straightforward protocol to perform quantitative analyses of both proteome and phosphoproteome from the same sample using iTRAQ.
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Otto-Hahn-Str. 6b, 44227, Dortmund, Germany.
authors.forename: Fiorella A
authors.initials: FA
authors.lastname: Solari
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Otto-Hahn-Str. 6b, 44227, Dortmund, Germany.
authors.forename: Laxmikanth
authors.initials: L
authors.lastname: Kollipara
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Otto-Hahn-Str. 6b, 44227, Dortmund, Germany.|Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, Scotland, UK.
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Otto-Hahn-Str. 6b, 44227, Dortmund, Germany. rene.zahedi@isas.de.
authors.forename: René P
authors.initials: RP
authors.lastname: Zahedi
mesh_terms: D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid; D006801:Humans; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D010750:Phosphoproteins; D020543:Proteome
keywords: Phosphopeptide enrichment; Quantitative phosphoproteomics; Quantitative proteomics; iTRAQ