(2006) Gene expression profiling of mouse embryonic stem cell subpopulations. differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells. Embryonic stem (ES) cells, which are derived from the inner cell mass of early blastocysts (1), are pluripotent and capable of self-renewal (2). Therefore, they hold promise for cell-based regenerative medicine (3). In recent years, significant progress has been made toward understanding the characteristics of ES cells, including uncovering some notable transcription factors, such as OCT4 (4, 5), SOX2 (6), and NANOG (7, 8). These key regulators are thought to be critical for the differentiation of ES cells because of their unique expression profiles and their essential functions in early development. High throughput, genome-wide studies have also discovered hundreds of genes that are probably critical for the self-renewal and pluripotency of ES cells (9, 10). In addition to the transcription of genes, the phenotype of ES cells was also found to be tightly regulated by the translation of proteins of which the active/inactive status is usually often determined by phosphorylation or other post-translational modifications (11). To obtain a more comprehensive understanding of the molecular mechanisms underlying ES cell pluripotency and differentiation, some transcriptome and proteome studies have been performed on human or mouse ES cells (12C14). Mass spectrometry (MS)-based proteomics provides the means to determine the large quantity, modification, localization, and conversation of proteins on a JTK12 large scale (15). Efforts at proteomics analysis have led to full-scale profiling of the proteome of mouse embryonic stem (mES) cells, resulting in the investigation of nearly 2000 proteins (16, 17). Other recent studies have reported the identification of 2389 and 5111 proteins in mES cells, including some previously undetected stem cell markers (18, 19). All Goserelin of the studies mentioned above provide new insights into ES cell protein expression profiling. Nevertheless, some studies indicate that numerous decisive events in cellular responses are mediated or decided not only by changes in protein large quantity but also by protein post-translational modifications (20). Among these protein modifications, the phosphorylation of proteins through kinase activity was reported to act as a critical regulator of ES cell function (21). The largest phosphoproteome analysis of human ES (hES)1 cells performed to date recognized 10,844 phosphorylation sites, which were detected via the combination of two peptide fragmentation methods (22). In addition, two groups recently identified additional phosphoproteomes and profiled their dynamic activities in hES cells (23, 24). However, several major difficulties remain unresolved, such as the low large quantity of phosphoproteins in general, dynamic site occupancy, and inherently poor fragmentation of phosphopeptides. In contrast with hES cells, a global view of the mES cell phosphoproteome has not yet been fully achieved. mES cells are the most important models that can be used to investigate the stemness characteristics of stem cells; therefore, comprehensive information around the phosphorylation says and specific phosphosites in the mES cell proteome will provide an important resource for stem cell research. In the current study, we attempted to profile a large phosphoproteome data set from mES cells to further explore their phosphorylation characteristics. Furthermore, we developed a Goserelin StemCell Project web site to retrieve and analyze the data from the study. This information provides an overview of phosphorylation in mES cells and should be a useful resource for the future understanding of the basic biology in mES cells. EXPERIMENTAL PROCEDURES mES Goserelin Cell Culture An E14.1 cell strain, a substrain of the E14 strain of ES cells derived from the blastocyst of a 129/Ola strain mouse, was purchased from ATCC. The cells.