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Embryonic Stem Cell Immunobiology

Volume 1029 of the series Methods in Molecular Biology pp 163-189

Date:

Combined Total Proteomic and Phosphoproteomic Analysis of Human Pluripotent Stem Cells

  • Junjie HouAffiliated withAdvanced Proteomics, Sanford|Burnham Medical Research Institute
  • , Brian T. D. TobeAffiliated withStem Cell Biology and Regenerative Medicine Program, Sanford|Burnham Medical Research Institute
  • , Frederick LoAffiliated withMuscle Development and Regeneration Program, Sanford|Burnham Medical Research Institute
  • , Justin D. BlethrowAffiliated withThermo Fisher Scientific
  • , Andrew M. CrainAffiliated withStem Cell Biology and Regenerative Medicine Program, Sanford-Burnham Medical Research Institute
  • , Dieter A. WolfAffiliated withCancer Center, Sanford|Burnham Medical Research Institute
  • , Evan Y. SnyderAffiliated withStem Cell Biology and Regenerative Medicine Program, Sanford|Burnham Medical Research Institute
  • , Ilyas SingecAffiliated withStem Cell Biology and Regenerative Medicine Program, Sanford|Burnham Medical Research Institute
  • , Laurence M. BrillAffiliated withAdvanced Proteomics, Sanford|Burnham Medical Research Institute

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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.

Key words

Embryonic stem cells (ESCs) Induced pluripotent stem cells (iPSCs) Mass spectrometry (MS) Multidimensional liquid chromatography (MDLC) Proteomics Phosphoproteomics Self-­renewal Pluripotency Protein phosphorylation Posttranslational modification (PTM)