title: Global phosphoproteome analysis on human HepG2 hepatocytes using reversed-phase diagonal LC.
abstract: We present a phosphoproteomics approach using diagonal RP chromatography as the basic isolation principle. Phosphopeptides present in a tryptic digest of total cellular lysates were first enriched by Fe3+-immobilized metal ion affinity chromatography. Further sorting of the phosphopeptides took place in three steps. First, the resulting peptide mixture was fractionated over reversed-phase chromatography. Second, peptides present in each fraction were treated with phosphatases. Third, the dephosphorylated peptides were then more hydrophobic and shifted towards a later elution interval from the contaminating non-phosphopeptides eluting at the same position as during the primary run. Since the phosphopeptides are isolated as their dephosphorylated form, additional proof for their original phosphorylation state was obtained by split-differential 16O-18O labeling. The method was validated with alpha-casein phosphopeptides and consecutively applied on HepG2 cells. We identified 190 phosphorylated peptides from 152 different proteins. This dataset includes 38 novel protein phosphorylation sites.
authors.affiliation: Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.forename: An
authors.initials: A
authors.lastname: Staes
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Damme
authors.forename: Sara
authors.initials: S
authors.lastname: De Groot
authors.forename: Koen
authors.initials: K
authors.lastname: Hugelier
authors.forename: Hans
authors.initials: H
authors.lastname: Demol
authors.forename: Lennart
authors.initials: L
authors.lastname: Martens
authors.forename: Marc
authors.initials: M
authors.lastname: Goethals
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D040901:Proteomics
chemical_list: D010750:Phosphoproteins