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• W2113087532 abstract "Embryonic stem cells are a unique cell population capable both of self-renewal and of differentiation into all tissues in the adult organism. Despite the central importance of these cells, little information is available regarding the intracellular signaling pathways that govern self-renewal or early steps in the differentiation program. Embryonic stem cell growth and differentiation correlates with kinase activities, but with the exception of the JAK/STAT3 pathway, the relevant substrates are unknown. To identify candidate phosphoproteins with potential relevance to embryonic stem cell differentiation, a systems biology approach was used. Proteins were purified using phosphoprotein affinity columns, then separated by two-dimensional gel electrophoresis, and detected by silver stain before being identified by tandem mass spectrometry. By comparing preparations from undifferentiated and differentiating mouse embryonic stem cells, a set of proteins was identified that exhibited altered post-translational modifications that correlated with differentiation state. Evidence for altered post-translational modification included altered gel mobility, altered recovery after affinity purification, and direct mass spectra evidence. Affymetrix microarray analysis indicated that gene expression levels of these same proteins had minimal variability over the same differentiation period. Bioinformatic annotations indicated that this set of proteins is enriched with chromatin remodeling, catabolic, and chaperone functions. This set of candidate phosphoprotein regulators of stem cell differentiation includes products of genes previously noted to be enriched in embryonic stem cells at the mRNA expression level as well as proteins not associated previously with stem cell differentiation status. Embryonic stem cells are a unique cell population capable both of self-renewal and of differentiation into all tissues in the adult organism. Despite the central importance of these cells, little information is available regarding the intracellular signaling pathways that govern self-renewal or early steps in the differentiation program. Embryonic stem cell growth and differentiation correlates with kinase activities, but with the exception of the JAK/STAT3 pathway, the relevant substrates are unknown. To identify candidate phosphoproteins with potential relevance to embryonic stem cell differentiation, a systems biology approach was used. Proteins were purified using phosphoprotein affinity columns, then separated by two-dimensional gel electrophoresis, and detected by silver stain before being identified by tandem mass spectrometry. By comparing preparations from undifferentiated and differentiating mouse embryonic stem cells, a set of proteins was identified that exhibited altered post-translational modifications that correlated with differentiation state. Evidence for altered post-translational modification included altered gel mobility, altered recovery after affinity purification, and direct mass spectra evidence. Affymetrix microarray analysis indicated that gene expression levels of these same proteins had minimal variability over the same differentiation period. Bioinformatic annotations indicated that this set of proteins is enriched with chromatin remodeling, catabolic, and chaperone functions. This set of candidate phosphoprotein regulators of stem cell differentiation includes products of genes previously noted to be enriched in embryonic stem cells at the mRNA expression level as well as proteins not associated previously with stem cell differentiation status. Embryonic stem cells (ESCs) 1The abbreviations used are: ESC, embryonic stem cell; 2D, two-dimensional; 2DGE, two-dimensional gel electrophoresis; ES, embryonic stem; EB, embryoid body; LIF, leukemia-inhibitory factor; TRIM28, tripartite motif protein 28; TEBP, telomerase-binding protein; HRE, hormone response element; cPGES, cytosolic prostaglandin E synthase. 1The abbreviations used are: ESC, embryonic stem cell; 2D, two-dimensional; 2DGE, two-dimensional gel electrophoresis; ES, embryonic stem; EB, embryoid body; LIF, leukemia-inhibitory factor; TRIM28, tripartite motif protein 28; TEBP, telomerase-binding protein; HRE, hormone response element; cPGES, cytosolic prostaglandin E synthase. were originally identified in mice and subsequently characterized from human tissue sources (1Evans M.J. Kaufman M.H. Establishment in culture of pluripotential cells from mouse embryos.Nature. 1981; 292: 154-156Google Scholar, 2Martin G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7634-7638Google Scholar, 3Thomson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshall V.S. Jones J.M. Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Google Scholar). The development of living mice wholly derived from such cells has demonstrated the pluripotency of murine embryonic stem cells (4Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Google Scholar). ESCs are capable of self-propagation by symmetrical division while retaining pluripotency. This phenotype is maintained by a combination of extrinsic and intrinsic factors (5Chambers I. Smith A. Self-renewal of teratocarcinoma and embryonic stem cells.Oncogene. 2004; 23: 7150-7160Google Scholar). The known extrinsic factors are leukemia-inhibitory factor (LIF) and bone morphogenetic proteins. LIF signaling through gp130 results in phosphorylation, dimerization, and nuclear translocation of the signal transducer and activator of transcription STAT3 (6Burdon T. Smith A. Savatier P. Signalling, cell cycle and pluripotency in embryonic stem cells.Trends Cell Biol. 2002; 12: 432-438Google Scholar). Bone morphogenetic protein treatment induces expression of Id family transcriptional modulators (5Chambers I. Smith A. Self-renewal of teratocarcinoma and embryonic stem cells.Oncogene. 2004; 23: 7150-7160Google Scholar). The principle known intrinsic factors that maintain the ESC phenotype are the transcription factor OCT-4 and the novel homeodomain protein Nanog. When ESCs are cultured in suspension in the absence of extrinsic factors, spherical multicellular structures termed embryoid bodies (EBs) are formed, and the individual cells comprising the sphere begin to differentiate along various lineages in a disorganized fashion (7Doetschman T.C. Eistetter H. Katz M. Schmidt W. Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium.J. Embryol. Exp. Morphol. 1985; 87: 27-45Google Scholar). Differentiation into multicellular spheres is also a property of other stem cells, such as the formation of neurospheres from neuronal stem cells. Control of the differentiation process is a topic of intense interest because of the potential application of stem cells to regenerate diseased or injured tissues. Apart from the small number of known intrinsic and extrinsic factors that maintain the embryonic stem cell phenotype, the molecular basis for the pluripotency and self-renewal of stem cells is not well understood. A number of studies have used RNA expression profiling techniques in an attempt to identify genes that specify stem cell properties in mice (8Ramalho-Santos M. Yoon S. Matsuzaki Y. Mulligan R.C. Melton D.A. “Stemness”: transcriptional profiling of embryonic and adult stem cells.Science. 2002; 298: 597-600Google Scholar, 9Ivanova N.B. Dimos J.T. Schaniel C. Hackney J.A. Moore K.A. Lemischka I.R. A stem cell molecular signature.Science. 2002; 298: 601-604Google Scholar, 10Perez-Iratxeta C. Palidwor G. Porter C.J. Sanche N.A. Huska M.R. Suomela B.P. Muro E.M. Krzyzanowski P.M. Hughes E. Campbell P.A. Rudnicki M.A. Andrade M.A. Study of stem cell function using microarray experiments.FEBS Lett. 2005; 579: 1795-1801Google Scholar) and humans (11Bhattacharya B. Miura T. Brandenberger R. Mejido J. Luo Y. Yang A.X. Joshi B.H. Ginis I. Thies R.S. Amit M. Lyons I. Condie B.G. Itskovitz-Eldor J. Rao M.S. Puri R.K. Gene expression in human embryonic stem cell lines: unique molecular signature.Blood. 2004; 103: 2956-2964Google Scholar, 12Abeyta M.J. Clark A.T. Rodriguez R.T. Bodnar M.S. Pera R.A. Firpo M.T. Unique gene expression signatures of independently-derived human embryonic stem cell lines.Hum. Mol. Genet. 2004; 13: 601-608Google Scholar, 13Richards M. Tan S.P. Tan J.H. Chan W.K. Bongso A. The transcriptome profile of human embryonic stem cells as defined by SAGE.Stem Cells. 2004; 22: 51-64Google Scholar). One group of studies revealed a set of 332 genes whose expression appears to be enriched in murine ESCs (8Ramalho-Santos M. Yoon S. Matsuzaki Y. Mulligan R.C. Melton D.A. “Stemness”: transcriptional profiling of embryonic and adult stem cells.Science. 2002; 298: 597-600Google Scholar, 9Ivanova N.B. Dimos J.T. Schaniel C. Hackney J.A. Moore K.A. Lemischka I.R. A stem cell molecular signature.Science. 2002; 298: 601-604Google Scholar, 14Fortunel N.O. Otu H.H. Ng H.H. Chen J. Mu X. Chevassut T. Li X. Joseph M. Bailey C. Hatzfeld J.A. Hatzfeld A. Usta F. Vega V.B. Long P.M. Libermann T.A. Lim B. Comment on “‘Stemness’: transcriptional profiling of embryonic and adult stem cells” and “a stem cell molecular signature”.Science. 2003; 302: 393Google Scholar). However, when data from hematopoietic and neuronal stem cells were included, cross-study comparison yielded almost no consensus, suggesting that the stem cell phenotype cannot be explained by transcriptional profile alone (14Fortunel N.O. Otu H.H. Ng H.H. Chen J. Mu X. Chevassut T. Li X. Joseph M. Bailey C. Hatzfeld J.A. Hatzfeld A. Usta F. Vega V.B. Long P.M. Libermann T.A. Lim B. Comment on “‘Stemness’: transcriptional profiling of embryonic and adult stem cells” and “a stem cell molecular signature”.Science. 2003; 302: 393Google Scholar, 15Evsikov A.V. Solter D. Comment on “‘Stemness’: transcriptional profiling of embryonic and adult stem cells” and “a stem cell molecular signature”.Science. 2003; 302: 393Google Scholar). Clearly mechanisms operating at the post-transcriptional level may also be relevant. To date, proteomic analysis of embryonic stem cells has been limited. Guo et al. (16Guo X. Ying W. Wan J. Hu Z. Qian X. Zhang H. He F. Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all-trans retinoic acid in vitro.Electrophoresis. 2001; 22: 3067-3075Google Scholar) used comparative two-dimensional gel electrophoresis (2DGE) to examine the differentiation of ESCs into neural cells under retinoic acid treatment and found 24 differentially expressed spots of which 12 were identified. Elliott et al. (17Elliott S.T. Crider D.G. Garham C.P. Boheler K.R. Van Eyk J.E. Two-dimensional gel electrophoresis database of murine R1 embryonic stem cells.Proteomics. 2004; 44032 Google Scholar) produced a two-dimension gel electrophoresis map of proteins in the mouse ESC line R1 and identified 218 proteins. Recently Nagano et al. (18Nagano K. Taoka M. Yamauchi Y. Itagaki C. Shinkawa T. Nunomura K. Okamura N. Takahashi N. Izumi T. Isobe T. Large-scale identification of proteins expressed in mouse embryonic stem cells.Proteomics. 2005; 5: 1346-1361Google Scholar) used 2D LC-MS/MS to identify ∼1800 proteins from the ESC line E14-1. 35 of these proteins yielded mass spectra that were consistent with phosphorylation, and one phosphorylation site was mapped. To specifically examine phosphorylation, Prudhomme et al. (19Prudhomme W. Daley G.Q. Zandstra P. Lauffenburger D.A. Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2900-2905Google Scholar) used quantitative Western blots to analyze how the phosphorylation status of 31 selected proteins correlated, in a combinatorial fashion, with mouse ESC proliferation or differentiation and found that the phosphorylation status of the kinases RAF1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, extracellular signal-regulated kinase, protein kinase B-α, SRC, and protein kinase C-ε were especially significant. As such, these data suggest that kinase activity and the phosphorylation status of target substrates may act as critical regulators of stem cell function. Here we sought to characterize the stem cell state by identifying the phosphoproteome associated with mouse ESCs and their derived EBs. Phosphoprotein enrichment and comparative 2DGE were used to profile phosphoprotein expression. Proteins detected by silver stain were identified by MALDI-MS/MS or by LC-MS/MS. The set of proteins that exhibited altered post-translational modification during differentiation included several proteins identified previously in gene expression arrays as conserved features of the stem cell phenotype. Proteins related to protein catabolism, protein folding, chromatin remodeling, and other functions were identified and found to exhibit evidence of altered phosphorylation between the ESC and EB states. The murine embryonic stem cell line J1 (20Li E. Bestor T.H. Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.Cell. 1992; 69: 915-926Google Scholar) was maintained on a feeder layer of murine embryonic fibroblasts. Prior to each experiment feeder cells were removed by briefly passaging on a gelatin-coated plate. A portion of the cells were differentiated into EBs by culturing for 24 h in bacteriological grade Petri dishes, which do not support ESC adherence. The remaining cells were plated on tissue culture dishes and maintained as ESC colonies in the presence of LIF for 24 h. The successful removal of feeder cells and formation of ESC colonies and EBs were verified by direct observation of cell morphology under light microscopy. Phosphoprotein enrichment was performed using Phosphoprotein Purification Kit 37101 (Qiagen, Mississauga, Ontario) as described previously (21Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Google Scholar). Briefly 2.5 mg of protein was loaded onto phosphoprotein binding columns and washed extensively before eluting bound proteins that were then concentrated using the supplied ultrafiltration columns (10-kDa cut-off). 2DGE was performed as described previously (21Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Google Scholar) using 8–16% gradient precast gels (Bio-Rad) or 10% hand-cast gels in a Protean II electrophoresis cell (Bio-Rad). For isoelectric focusing (first separation dimension), three different pH ranges were used: 3–10 (done twice), 5–8 (done once), and 4–7 (done twice). Each experiment was done as an ES versus EB comparison pair for a total of 10 two-dimensional gels. Silver staining was performed by a standard method (22Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Google Scholar). For LC-MS/MS, samples were prepared as described previously (21Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Google Scholar). For MALDI-MS/MS samples, the in-gel digest was performed manually and extracted in 5% formic acid. Prior to spotting for MALDI analysis, samples were cleaned using ZipTips (Millipore, Billerica, MA) following the manufacturer’s recommended procedure. Nano-LC-MS/MS was performed as described previously (21Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Google Scholar) using an Ultima Q-TOF hybrid tandem mass spectrometer (Waters). MALDI-MS/MS spectra were acquired using a QSTAR XL tandem mass spectrometer (ABI/Sciex) with an oMALDI-2 source and Analyst QS version 1.1, build 9865. α-Cyano-4-hydroxycinnamic acid (Agilent, Palo Alto, CA) matrix was used. Spectra were searched against the National Center for Biotechnology Information non-redundant 20050606 database using Mascot daemon version 2.0.5 on an in-house Mascot server version 2.0.04. Parameters used for queries were trypsin cleavage, two missed cleavages, ±1.2-Da peptide tolerance, ±0.6-Da MS/MS tolerance, and the following variable modifications: acetyl (N-terminal), carbamidomethyl (Cys), deamidation (Asn, Glu), oxidation (Met), phosphorylation (Thr, Ser, Tyr), and pyro-Glu (N-terminal Glu). The results were then evaluated manually, and search parameters were narrowed as warranted to eliminate potential false-positive identifications. To profile the phosphoproteome of ESCs and to identify proteins with potential relevance to ESC differentiation, phosphoprotein enrichment and comparative two-dimensional gel electrophoresis were performed on ESC and EB samples before identification of proteins by MS/MS. Cell lysates were passed over phosphoprotein affinity columns, and eluting proteins were detected by 2DGE and silver stain (Fig. 1). As expected for phosphoproteins (21Puente L.G. Carriere J.F. Kelly J.F. Megeney L.A. Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.FEBS Lett. 2004; 574: 138-144Google Scholar), most spots were present at low isoelectric points in the pH 4–6 range (Fig. 1). Including both ESC and EB samples, a total of 1367 protein spots were detected over 10 gels. Within each pair of gels, the majority of spots (∼80%) were present at equal apparent abundance and gel mobility when ESC and EB were compared. In total 283 spots exhibited obvious changes in intensity (estimated change of at least 30%) or mobility when pairs of ESC and EB samples were compared. From the gels, 362 spots were excised, giving preference to spots that appeared to be differentially expressed between ESC and EB. 332 identifications were made (Table I) that represented 108 different proteins (many proteins were independently identified multiple times). 30 proteins gave rise to multiple mobility species, and a total of 183 protein species were observed (Table I).Table IProteins identified from embryonic stem cells and embryoid bodies after phosphoprotein enrichmentView Large Image Figure ViewerDownload (PPT)View Large Image Figure ViewerDownload (PPT)View Large Image Figure ViewerDownload (PPT) Open table in a new tab Phosphopeptide mass spectra are rarely observed in protein identification experiments because of the rarity of phosphopeptides among total peptides and because of the very low ionization efficiency of phosphorylated peptides in positive ion mode (23Janek K. Wenschuh H. Bienert M. Krause E. Phosphopeptide analysis by positive and negative ion matrix-assisted laser desorption/ionization mass spectrometry.Rapid Commun. Mass Spectrom. 2001; 15: 1593-1599Google Scholar). However in our study, after phosphoprotein affinity treatment, we observed mass spectra that were consistent with phosphorylation for several proteins: HSPB1, p23/TEBP, CKAP1/TBCB, stathmin, hepatoma-derived growth factor, and cofilin. In four cases, specific sites of phosphorylation were mapped (Fig. 2). In stathmin, the phosphopeptides ESVPDFPLpSPPK (where pS is phosphoserine) (Ser-37) and ASGQAFELILpSPR (Ser-24) were observed (Fig. 2, A and B) and represent previously known sites of phosphorylation (24Beretta L. Dobransky T. Sobel A. Multiple phosphorylation of stathmin. Identification of four sites phosphorylated in intact cells and in vitro by cyclic AMP-dependent protein kinase and p34cdc2.J. Biol. Chem. 1993; 268: 20076-20084Google Scholar). Two gel mobility species of stathmin were observed of which only the lower pI form showed Ser-24 phosphorylation. Both phosphorylated and unphosphorylated forms of the HSPB1 tryptic peptide SPSWEPFR were observed with a +80 difference in mass/charge characteristic of phosphorylation (Fig. 2C). The MS/MS spectrum of the HSPB1 phosphopeptide was consistent with phosphorylation at Ser-13 (not shown). In other cell types, this peptide can be phosphorylated at multiple sites including Ser-15 (25Lee Y.J. Lee D.H. Cho C.K. Bae S. Jhon G.J. Lee S.J. Soh J.W. Lee Y.S. HSP25 inhibits protein kinase Cδ-mediated cell death through direct interaction.J. Biol. Chem. 2005; 280: 18108-18119Google Scholar). For cofilin, the peptide N-acetyl-ApSGVAVSDGVIK was observed (Fig. 2D) that matches the known biochemistry of this protein (26Bamburg J.R. Proteins of the ADF/cofilin family: essential regulators of actin dynamics.Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Google Scholar). A key goal of our study was to identify candidate regulators of stem cell differentiation by identifying proteins whose phosphorylation status is altered in a manner that correlates with ESC differentiation status. Following phosphoprotein affinity column treatment and 2DGE, protein identities and silver stain patterns were carefully correlated across the gels. For each identified spot on each pair of gels, silver staining was assessed as either greater in ESC, greater in EB, or unchanged (marked as symbols in Table I). Only strong alterations (∼30% or greater increase or decrease) in silver stain intensity were scored as changes so that across all samples only 20% of spots were considered to exhibit change. 20 protein species were identified that were repeatedly detected more strongly in EB, whereas 15 species were repeatedly observed preferentially or exclusively in ES samples (Table II). 11 cases were identified in which a protein was present at different electrophoretic mobilities when ESC and EB were compared (Table II).Table IIProteins exhibiting differential post-translational modifications between ESC and EBA. Protein species with increased detection in ESCsCbx3, Ckap/Tbcb, Cln1a/pICLn*, Hdgf, Hspca*, Hspcb, Psma3, Ptma, Ranbp1, Tebp/cPGEs, Tpm2, Trim28a, Trim28-related fragmentB. Protein species with increased detection in EBsAnp32a, Anp32b, Ascl1, Calm1, Calr, Cdc37, DN38/Nap111, eIF3 s2, Habp/C1qbp, Hmgb2, Hspb1, Hspca, Npm1, Psma2, Psmc2, Psmc5/Trip1, Psmc6, Stip1*, TubulinC. Evidence of altered gel mobility in ESC vs. EBAnp32a, Anp32b, Anp32e, eIF2b2, Habp1/C1qbp, Hspca, Hspcb, Psma3, Psmb6, SET, Tra1 Open table in a new tab An important question in the interpretation of the phosphoprotein enrichment screen is whether lack of detection of a protein under a given condition reflects a lack of phosphorylation or a lack of expression. Therefore, we examined gene expression profiles for our proteins of interest. The J1 ES cell line has been extensively characterized with respect to gene expression using Affymetrix™ gene arrays (10Perez-Iratxeta C. Palidwor G. Porter C.J. Sanche N.A. Huska M.R. Suomela B.P. Muro E.M. Krzyzanowski P.M. Hughes E. Campbell P.A. Rudnicki M.A. Andrade M.A. Study of stem cell function using microarray experiments.FEBS Lett. 2005; 579: 1795-1801Google Scholar). Gene array data for 0, 6, 12, 18, and 24 h of J1 ES cell differentiation was extracted from the StemBase (10Perez-Iratxeta C. Palidwor G. Porter C.J. Sanche N.A. Huska M.R. Suomela B.P. Muro E.M. Krzyzanowski P.M. Hughes E. Campbell P.A. Rudnicki M.A. Andrade M.A. Study of stem cell function using microarray experiments.FEBS Lett. 2005; 579: 1795-1801Google Scholar) database (the 0- and 24-h samples in the database correspond to our ESC and EB samples, respectively). With the exception of ASCL1, every protein listed in Table II matched to one or more Affymetrix probe sets that were classed as “Present” in J1 ES cells. Gene expression data were also examined quantitatively. For each probe that matched to a protein of interest, gene chip average signal (proportional to gene expression) at 24 h of EB formation was plotted against average signal at 0 h (i.e. non-differentiated ESC) (Fig. 3). In most cases deviations from the diagonal were minimal (Fig. 3). All 24-h average signal values fell between 0.5 and 1.5 times the time 0 value. These observations support the interpretation that the proteins listed in Table II are similarly expressed in both ESC and EB but are differentially phosphorylated between the two conditions. To gain insight into the putative biological functions of the identified proteins, Gene Ontology annotations were extracted from the Mouse Genome Database (27Blake J.A. Richardson J.E. Bult C.J. Kadin J.A. Eppig J.T. MGD: the Mouse Genome Database.Nucleic Acids Res. 2003; 31: 193-195Google Scholar). For the set of proteins we identified as being expressed in J1 ES cells (a subset of Table I), the most common Biological Process annotations were heat shock/chaperone, protein catabolism, protein biosynthesis, and cytoskeleton organization (Fig. 4, inner pie chart). For the set of proteins that exhibited changes in post-translational modification when undifferentiated and differentiated cells were compared (Table II), the corresponding biological process annotations were even further enriched in heat shock and protein catabolism functions as well as transcription and chromatin modulating functions (Fig. 4, outer pie chart), whereas annotations related to protein biosynthesis, cytoskeleton organization, and other functions were less abundant. After 2DGE, peptides related to TRIM28 (TIF1β) were detected in two distinct gel mobility species. One species was a high molecular mass spot that contained multiple peptides that were matched to various regions of the 90-kDa TRIM28 sequence by MS/MS data (Fig. 5). A second species of ∼17-kDa was identified in two independent experiments that contained the peptides ADVQSIIGLQR and LSPPYSSPQEFAQDVGR. These peptide sequences are known to occur in only two cases: TRIM28 and unnamed protein product gi|26354228 whose sequence is identical to the C-terminal 240 amino acids of TRIM28 (predicted mass, 26 kDa). These data demonstrate that a protein product identical to or smaller than the predicted protein gi|26354228 and containing a sequence identical to the TRIM28 BROMO domain is present in mouse ESCs. By examining the phosphoprotein-enriched proteome of mouse ESC and EB, 108 proteins and 183 protein species were identified (Table I). Importantly 39 proteins (46 protein species) were identified that reproducibly exhibited distinct patterns of post-translational modification (as indicated by affinity for phosphoprotein binding columns and/or altered electrophoretic gel mobility) between the undifferentiated ESC and differentiating EB states (Table II). Such observations are correlative but nevertheless suggest these proteins may be functionally linked to ESC differentiation. Current phosphoprotein profiling methods are by no means comprehensive, and the candidates identified here undoubtedly represent only a subset of the complete phosphoproteome. An additional method to address sample complexity is by using HPLC separation technology in concert with mass spectrometry (18Nagano K. Taoka M. Yamauchi Y. Itagaki C. Shinkawa T. Nunomura K. Okamura N. Takahashi N. Izumi T. Isobe T. Large-scale identification of proteins expressed in mouse embryonic stem cells.Proteomics. 2005; 5: 1346-1361Google Scholar). Indeed in one experiment analyzed by LC-MS/MS we noted 51 additional protein species that were not seen in the MALDI-MS/MS experiments (this accounts for a substantial portion of the apparent experiment-to-experiment variability seen in Table I). In practical terms, however, MALDI-MS/MS was found to offer a significant savings in terms of time required for processing large numbers of samples. Phosphoprotein profiling techniques that dispense with 2D gels and use only HPLC separation can achieve high proteome coverage (18Nagano K. Taoka M. Yamauchi Y. Itagaki C. Shinkawa T. Nunomura K. Okamura N. Takahashi N. Izumi T. Isobe T. Large-scale identification of proteins expressed in mouse embryonic stem cells.Proteomics. 2005; 5: 1346-1361Google Scholar). However, in our study 108 different proteins produced 183 gel mobility species indicating that a large amount of protein modification data may be overlooked in gel-free experiments. Two previous studies have examined the general proteome of ES cells (17, 18). 70 of the proteins identified in our study were not identified by Elliott et al. (17Elliott S.T. Crider D.G. Garham C.P. Boheler K.R. Van Eyk J.E. Two-dimensional gel electrophoresis database of murine R1 embryonic stem cells.Proteomics. 2004; 44032 Google Scholar), consistent with the interpretation that the phosphoprotein enrichment method selectively captures a less abundant subset of the proteome. Nagano et al. (18Nagano K. Taoka M. Yamauchi Y. Itagaki C. Shinkawa T. Nunomura K. Okamura N. Takahashi N. Izumi T. Isobe T. Large-scale identification of proteins expressed in mouse embryonic stem cells.Proteomics. 2005; 5: 1346-1361Google Scholar) identified over 1700 proteins from E14-1 cells of which 35 had features that suggested potential phosphorylat" @default.
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• W2113087532 date "2006-01-01" @default.
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• W2113087532 title "Identification of Candidate Regulators of Embryonic Stem Cell Differentiation by Comparative Phosphoprotein Affinity Profiling" @default.
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• W2113087532 doi "https://doi.org/10.1074/mcp.m500166-mcp200" @default.
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