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.
