title: Phospho-iTRAQ: assessing isobaric labels for the large-scale study of phosphopeptide stoichiometry.
abstract: The ability to distinguish between phosphopeptides of high and low stoichiometry is essential to discover the true extent of protein phosphorylation. We here extend the strategy whereby a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part. Our use of isobaric tags for relative and absolute quantitation (iTRAQ) marks the first time that isobaric tags have been applied for the large-scale analysis of phosphopeptides. Our Phospho-iTRAQ method focuses on the unmodified counterparts of phosphorylated peptides, which thus circumvents the ionization, fragmentation, and phospho-enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. Since iTRAQ enables multiplexing, simultaneous (phospho)proteome comparison between internal replicates and multiple samples is possible. The technique was validated on multiple instrument platforms by adding internal standards of high stoichiometry to a complex lysate of control and EGF-stimulated HeLa cells. To demonstrate the flexibility of Phospho-iTRAQ with regards to the experimental setup, the proteome coverage was extended through gel fractionation, while an internal replicate measurement created more stringent data analysis opportunities. The latest developments in MS instrumentation promise to further increase the resolution of the stoichiometric measurement of Phospho-iTRAQ in the future. The data have been deposited to the ProteomeXchange with identifier PXD001574.
authors.affiliation: Laboratory of Pharmaceutical Biotechnology and ‡Department of Biochemistry, Ghent University , B-9000 Ghent, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Glibert
authors.forename: Paulien
authors.initials: P
authors.lastname: Meert
authors.forename: Katleen
authors.initials: K
authors.lastname: Van Steendam
authors.forename: Filip
authors.initials: F
authors.lastname: Van Nieuwerburgh
authors.forename: Dieter
authors.initials: D
authors.lastname: De Coninck
authors.forename: Lennart
authors.initials: L
authors.lastname: Martens
authors.forename: Maarten
authors.initials: M
authors.lastname: Dhaenens
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
mesh_terms: D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D010748:Phosphopeptides
keywords: dephosphotylation; iTRAQ; in-gel Phospho-iTRAQ; phosphoproteome; stoichomtery; threshold calculation