title: Coupled global and targeted proteomics of human embryonic stem cells during induced differentiation.
abstract: Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomics studies. The current investigation avoided the use of MEFs or MEF-conditioned medium for hESC culture, allowing global proteomics analysis without these confounding conditions, and elucidated neural cell-specific signaling pathways involved in noggin-induced hESC differentiation. Based on these analyses, we propose the following early markers of hESC neural differentiation: collapsin response mediator proteins 2 and 4 and the nuclear autoantigenic sperm protein as a marker of pluripotent hESCs. We then developed a directed mass spectrometry assay using multiple reaction monitoring (MRM) to identify and quantify these markers and in addition the epidermal ectoderm marker cytokeratin-8. Analysis of global proteomics, quantitative RT-PCR, and MRM data led to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show that targeted MRM analysis on non-redundant peptides provides more exact quantification of homologous proteins. This study describes the facile transition from discovery proteomics to targeted MRM analysis and allowed us to identify and verify several potential biomarkers for hESCs during noggin-induced neural and BMP4-induced epidermal ectoderm differentiation.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA. akyocum@umich.edu
authors.forename: Anastasia K
authors.initials: AK
authors.lastname: Yocum
authors.forename: Theresa E
authors.initials: TE
authors.lastname: Gratsch
authors.forename: Nancy
authors.initials: N
authors.lastname: Leff
authors.forename: John R
authors.initials: JR
authors.lastname: Strahler
authors.forename: Christie L
authors.initials: CL
authors.lastname: Hunter
authors.forename: Angela K
authors.initials: AK
authors.lastname: Walker
authors.forename: George
authors.initials: G
authors.lastname: Michailidis
authors.forename: Gilbert S
authors.initials: GS
authors.lastname: Omenn
authors.forename: K Sue
authors.initials: KS
authors.lastname: O'Shea
authors.forename: Philip C
authors.initials: PC
authors.lastname: Andrews
mesh_terms: D000595:Amino Acid Sequence; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000078404:Epidermal Cells; D004817:Epidermis / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D000099176:Noggin Protein
chemical_list: D015415:Biomarkers; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D002352:Carrier Proteins; D010455:Peptides; D011506:Proteins; D012333:RNA, Messenger; D000099176:Noggin Protein; C525809:BMP4 protein, human; C525810:Bmp4 protein, mouse