Abstract
Despite advances in understanding pluripotency through traditional cell biology and gene expression profiling, the signaling networks responsible for maintenance of pluripotency and lineage-specific differentiation are poorly defined. To aid in an improved understanding of these networks at the systems level, we present procedures for the combined analysis of the total proteome and total phosphoproteome (termed (phospho)proteome) from human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), and their differentiated derivatives. Because there has been considerable heterogeneity in the literature on the culture of pluripotent cells, we first briefly describe our feeder-free cell culture protocol. The focus, however, is on procedures necessary to generate large-scale (phospho)proteomic data from the cells. Human cells are described here, but the (phospho)proteomic procedures are broadly applicable. Detailed procedures are given for lysis of the cells, protein sample preparation and digestion, multidimensional liquid chromatography, analysis by tandem mass spectrometry, and database searches for peptide/protein identification (ID). We summarize additional data analysis procedures, the subject of ongoing efforts.
Authors Junjie Hou and Brian T.D. Tope contributed equally to this chapter.
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Acknowledgments
We thank Anjum Khan and Dante Bencivengo of Thermo Fisher Scientific; Peter Kent, Lori Ann Upton, David Mintline, and Kerry Nugent of Michrom Bioresources for support with instrumentation; David Chiang and Patrick Chu from SageN and Kutbuddin Doctor from the Sanford-Burnham Medical Research Institute (SBMRI) for bioinformatics support; as well as Khatereh Motamedchaboki and Wenhong Zhu from SBMRI for help with some of the experiments. Support was provided by the SBMRI, NCI Cancer Center Support Grant 5 P30 CA30199-28, The La Jolla Interdisciplinary Neuroscience Center Cores Grant 5 P30 NS057096 from NINDS, and RC2 MH090011 from NIMH.
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Hou, J. et al. (2013). Combined Total Proteomic and Phosphoproteomic Analysis of Human Pluripotent Stem Cells. In: Zavazava, N. (eds) Embryonic Stem Cell Immunobiology. Methods in Molecular Biology, vol 1029. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-478-4_12
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DOI: https://doi.org/10.1007/978-1-62703-478-4_12
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