title: Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry.
abstract: Protein phosphorylation is central to the understanding of cellular signaling, and cellular signaling is suggested to play a major role in the regulation of human embryonic stem (ES) cell pluripotency. Here, we describe the use of conventional tandem mass spectrometry-based sequencing technology--collision-activated dissociation (CAD)--and the more recently developed method electron transfer dissociation (ETD) to characterize the human ES cell phosphoproteome. In total, these experiments resulted in the identification of 11,995 unique phosphopeptides, corresponding to 10,844 nonredundant phosphorylation sites, at a 1% false discovery rate (FDR). Among these phosphorylation sites are 5 localized to 2 pluripotency critical transcription factors--OCT4 and SOX2. From these experiments, we conclude that ETD identifies a larger number of unique phosphopeptides than CAD (8,087 to 3,868), more frequently localizes the phosphorylation site to a specific residue (49.8% compared with 29.6%), and sequences whole classes of phosphopeptides previously unobserved.
authors.affiliation: Department of Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
authors.forename: Danielle L
authors.initials: DL
authors.lastname: Swaney
authors.forename: Craig D
authors.initials: CD
authors.lastname: Wenger
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids; D002478:Cells, Cultured; D004583:Electrons; D053595:Embryonic Stem Cells / Q000378:metabolism*; D006801:Humans; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome