Phosphoproteomic Analysis of Human Embryonic Stem Cells

Abstract

SUMMARY Protein phosphorylation, while critical to cellular behavior, has been under-characterized in pluripotent cells. Therefore, we performed phosphoproteomic analyses of human embryonic stem cells (hESCs) and their differentiated derivatives. 2546 phosphorylation sites were identified on 1602 phosphoproteins; 389 proteins contained more phosphorylation site identifications in undifferentiated hESCs, whereas 540 contained more such identifications in differentiated derivatives. Phosphoproteins in receptor tyrosine kinase (RTK) signaling pathways were numerous in undifferentiated hESCs. Cellular assays corroborated this observation by showing that multiple RTKs cooperatively supported undifferentiated hESCs. In addition to bFGF, EGFR, VEGFR and PDGFR activation was critical to the undifferentiated state of hESCs. PDGF-AA complemented a sub-threshold bFGF concentration to maintain undifferentiated hESCs. Also consistent with phosphoproteomics, JNK activity participated in maintenance of undifferentiated hESCs. These results support the utility of phosphoproteomic data, provide guidance for investigating known and novel proteins in hESCs, and complement transcriptomics/epigenetics for broadening our understanding of hESC fate determination.

Temporal changes in Hec1 phosphorylation control kinetochore–microtubule attachment stability during mitosis

“…S5). Given the published proteomic studies on Ser69 (Brill et al, 2009;Malik et al, 2009;Olsen et al, 2010), it will be interesting in the future to determine whether the temporal regulation of Ser69 phosphorylation is similar to that of the phosphorylated amino acids described here.…”

“…Previous proteomics studies using isolated mitotic HeLa cell spindles or human embryonic stem cells have demonstrated that Hec1 is phosphorylated on Ser55 and Ser69 in vivo by undetermined kinases (Nousiainen et al, 2006;Malik et al, 2009;Brill et al, 2009;Olsen et al, 2010). We now include Ser44, Ser15 and Ser8 to the list of known in vivo N-terminal Hec1 phosphorylation sites (supplementary material Fig.…”

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

“…The most commonly used affinity-based methods are the (Fe 3+ )-based immobilized metal affinity chromatography (Fe 3+ -IMAC) [6,21,[26][27][28] and titanium dioxide (TiO 2 ) affinity chromatography [9,14,29,30]. Recently, on the basis of these methods, some new enrichment strategies have been developed by adopting different metal oxides (ZrO 2 and Nb 2 O 5 ) or IMAC with alternative metal ions (Ga 3+ , Zr 4+ , and Ti 4+ ) [19,20].…”

Modeling heterogeneity in the pluripotent state: A promising strategy for improving the efficiency and fidelity of stem cell differentiation

“…open/close chromatin regions, and activating/inhibitory chromatin marks patterns 25, 26, 32, 33. Moreover, although it is not yet possible to perform single cell measurements of signaling pathways, the inclusion of phosphoproteomics data in these network models will include another layer of information for contextualizing network interactions in this layer, depending on the post‐translational modifications determining the activity of signalling proteins 105, 106.…”