title: Quantitative proteome and phosphoproteome analysis of human pluripotent stem cells.
abstract: Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite for better controlling stem cell differentiation to specific fates. Reversible protein phosphorylation is one of the most important posttranslational modifications regulating signaling pathways in biological processes. Global analysis of dynamic changes in protein phosphorylation is, therefore, key to understanding signaling at the system level. Here, we describe a generic mass spectrometry (MS)-based phosphoproteomics strategy applied to monitor phosphorylation dynamics after bone morphogenetic protein 4 (BMP4)-induced differentiation of human embryonic stem cells (hESCs). Our method combines the use of strong cation exchange (SCX) and titanium dioxide (TiO(2)) for phosphopeptide enrichment, high-resolution MS for peptide and protein identification, and stable isotope labeling by amino acids in cell culture (SILAC) for quantification. This approach allows us to identify thousands of phosphorylation sites and profile their relative abundance during differentiation. This systems-biology-based approach provides new insights into how human pluripotent stem cells exit the pluripotent state.
authors.affiliation: Bijvoet Center for Biomolecular Research, Biomolecular Mass Spectrometry and Proteomics Group, Utrecht, The Netherlands.
authors.forename: Javier
authors.initials: J
authors.lastname: Muñoz
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D019295:Computational Biology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007474:Ion Exchange; D007553:Isotope Labeling; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D055748:SOXB1 Transcription Factors / Q000737:chemistry / Q000378:metabolism; D012984:Software; D012996:Solutions
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D012996:Solutions