Table 1. Phosphopeptides identified as changing in differentiating ES on analysis of iTRAQTM relative quantification.
| Acc.nb | Gene name | Protein name | Phosphopeptide sequence | Bry+Flk-: Bry-Flk- | Bry+Flk+: Bry+Flk− | Bry+Flk+: Bry−Flk− | GO Biological Process | GO Molecular Function |
|---|---|---|---|---|---|---|---|---|
| P57776 | Eef1d | eEF1d protein | GATPAEDDE DKDIDLFG pSDEEEEDKEAAR | 1.5 | 1.09 | 1.61 | Positive regulation of NF-kB cascade | Signal transducer activity; Translational elongation factor activity |
| P57776 | Eef1d | eEF1d protein | ATAPQTQ HVpSPMR | 1.96 | 0.77 | 1.48 | Positive regulation of NF-kB cascade | Signal transducer activity; Translational elongation factor activity |
| Q6J1H4 | Utf1 | UTF1 | RLPAFSPP SPApSPDAELR | 0.36 | 0.5 | 0.17 | Transcription, DNA-dependent | Transcription coactivator activity |
| Q6J1H4 | Utf1 | UTF1 | RLPAFpSPPS PApSPDAELR | 0.38 | 0.42 | 0.16 | Transcription, DNA-dependent | Transcription coactivator activity |
| Q6J1H4 | Utf1 | UTF1 | RLPAFSPP pSPASPDAELR | 0.37 | 0.54 | 0.2 | Transcription, DNA-dependent | Transcription coactivator activity |
| Q6J1H4 | Utf1 | UTF1 | RLPAFSPP pSPApSPDAELR | 0.31 | 0.58 | 0.18 | Transcription, DNA-dependent | Transcription coactivator activity |
| Q6J1H4 | Utf1 | UTF1 | SAGDVPVTTS DAFATSGGM AEPGpSPK | 0.39 | 0.48 | 0.19 | Transcription, DNA-dependent | Transcription coactivator activity |
| P17095 | Hmga1 | High mobility group protein HMG-I/HMG-Y | KQPPV pSPGTALVGSQK | 1.22 | 0.59 | 0.73 | Regulation of transcription; DNA-dependent, Negative regulation of cell proliferation | DNA-binding; Transcription coactivator activity |
| P52927 | Hmga2 | High mobility group protein HMG-A2 | KQQQEPTCEP pSPKRPR | 1.29 | 1.89 | 2.38 | Positive regulation of stem cell proliferation | A-T DNA binding |
| Q9CQS8 | Sec61b | Sec61 beta subunit | PGPTPSGTNVGS pSGRSPSK | 1.07 | 1.61 | 1.71 | Protein transport | Ribosome binding |
| Q9CQS8 | Sec61b | Sec61 beta subunit | PGPTPSGTNV GSSGRpSPSK | 1.1 | 1.52 | 1.63 | Protein transport | Ribosome binding |
| P30999 | Ctnnd1 | Catenin delta-1 (p120 catenin) | GSLApSLDSLRK | 0.78 | 0.62 | 0.48 | Wnt receptor signaling pathway; Regulation of transcription, DNA-dependent | Protein phosphatase binding; Protein kinase binding |
| Q8K019 | Bclaf1 | Bcl-2-associated transcription factor | IDIpSPSALRK | 0.53 | 1.35 | 0.7 | Regulation of transcription, DNA-dependent; Positive regulation of apoptosis | Protein binding; DNA binding |
| Q9WV60 | Gsk3b | Glycogen synthase kinase 3b | GEPNVpSYICSR | 0.15 | 1.48 | 1.67 | Genetic imprinting; Multicellular organismal development; Wnt receptor signaling pathway | Protein kinase binding; Transferase activity |
| O88509 | Dnmt3b | DNA (cytosine-5) methyltrans-ferase 3B | TTNDSAASE pSPPPKR | 0.38 | 0.51 | 0.19 | Methylation: Epigenetic | Transferase activity |
| Q01320 | Top2a | Topoisomerase (DNA) II alpha | KPIKYLEE pSDDDDDLF | 0.52 | 1.58 | 0.81 | ATP catabolic process; Mitotic recombination; Embryonic cleavage | DNA topoisomerase activity; Chromatin binding |
| P14733 | Lmnb1 | Lamin B1 | LKLpSPSPSSR | 0.64 | 0.77 | 0.48 | G2/M-specific positive regulation of cyclin-dependent protein kinase activity Positive regulation of JNK cascade | JUN kinase binding; Phospholipase binding |
| Q8BTI8 | Srrm2 | Serine/arginine repetitive matrix 2 | MVQASSQSLLP PAQDRPRp SPVPSAFSDQSR | 0.59 | 0.78 | 0.46 | RNA splicing; mRNA processing | Protein N-terminus binding |
| Q569Z6 | Thrap3 | Thyroid hormone receptor associated protein 3 | RIDIpSPSTFR | 0.56 | 1 | 0.55 | Steroid hormone receptor signaling pathway; Transcription - DNA dependent | Transcription coactivator activity; Nucleotide binding |
| P97496 | Smarcc1 | SWI/SNF complex 155 kDa subunit | RKPpSP pSPPPPTATESR | 1.6 | 0.75 | 1.2 | Chromatin remodeling; Organ morphogenesis; Transcription, DNA-dependent | Protein binding; DNA binding |
| Q8VDF2 | Uhrf1 | E3 ubiquitin-protein ligase UHRF1 | RPLIA pSPSQPPPALR | 0.82 | 0.7 | 0.56 | Cell proliferation; Regulation of transcription, DNA-dependent; Multicellular organismal development | DNA binding; Methyl-CpG binding |
| P60904 | Dnajc5 | DnaJ (Hsp40) homolog, subfamily C, member 5 | SLpSTSGESL YHVLGLDK | 1.35 | 1.11 | 1.51 | Protein folding; Negative regulation of neuron apoptosis | ATP-dependent protein binding; Heat shock protein binding; Unfolded protein binding |
| Q8K310 | Matr3 | Matrin-3 | TEpSPAE GKEQEEK | 0.49 | 1.16 | 0.58 | Unannotated | Nucleic acid binding |
| Q9Z1Q9 | Vars | Valyl-tRNA synthetase | LpSATVTEAF VRLHEEGVI | 1.44 | 0.56 | 0.79 | tRNA aminoacylation for protein translation | Aminoacyl-tRNA ligase activity; Nucleotide binding |
| Q80XU3 | Nucks1 | Nuclear ubiquitous casein and cdk substrate | ATVTP pSPVKGK | 0.56 | 0.85 | 0.47 | Unannotated | DNA binding |
| Q6PR54 | Rif1 | Rap1-interacting factor 1 | SSDpSVD IEEQEEK | 0.69 | 0.58 | 0.37 | Stem cell maintenance; cell cycle; Response to DNA damage stimulus | Protein binding |
| Q6PR54 | Rif1 | Rap1-interacting factor 1 | VSDSSL pSPEK | 0.84 | 0.64 | 0.53 | Stem cell maintenance; cell cycle; Response to DNA damage stimulus | Protein binding |
| Q8CBW3 | Abi1 | Abl interactor 1 | TNPPTQKPP SPPVpSGR | 0.99 | 1.67 | 1.64 | Peptidyl-tyrosine phosphorylation; Cell motility | Protein binding; Protein tyrosine kinase activator activity |
| Q08943 | Ssrp1 | FACT complex subunit SSRP1 | EGINPGYD DYADpSDE DQHDAYLER | 1.56 | 0.68 | 1.05 | Regulation of transcription, DNA-dependent | DNA binding |
| Q64012 | Raly | hnRNP-associated with lethal yellow | GRLpSPVPVPR | 2.53 | 0.47 | 1.37 | RNA splicing; mRNA processing | RNA binding |
| Q9JIX8 | Acin1 | Acinus | HLpSHPEP EQQHVIQR | 1.15 | 0.52 | 0.59 | Apoptosis; Apoptotic chromosome condensation | Nucleic acid binding; Nucleotide binding |
| P11499 | Hsp 90ab1 | Heat-shock protein hsp84 | IEDVGpSDE EDDSGKDK | 0.55 | 1.35 | 0.73 | Protein folding; Response to stress; Placenta development | Protein binding |
| P42208 | Sept2 | Septin 2 (NEDD5 protein) | IYHLPDAE pSDEDE DFKEQTR | 1.01 | 1.68 | 1.69 | Cell cycle | GTPase activity; Protein binding |
| O54839 | Eomes | Eomes | KGpSPC AEEELPS AATAAATAR | 1.52 | 0.76 | 1.15 | Blastocyst development; Multicellular organismal development; Stem cell maintenance; Mesoderm formation; Endoderm formation; Interferon-gamma production | DNA binding; Transcription factor activity |
| Q9CW46 | Raver1 | Ribonucleoprotein PTB-binding 1 | LLpSPIASNR | 1.33 | 0.49 | 0.65 | Unannotated | RNA binding |
| Q6P4S8 | Ints1 | Integrator complex subunit 1 | LSpSTP PLSALGR | 2.45 | 0.66 | 1.62 | snRNA processing; Blastocyst growth; Inner cell mass cell proliferation; Apoptosis | Unannotated |
| Q8CGU3 | Pnn | Pinin | RGFpSD SGGGPPAK | 1.26 | 1.37 | 1.71 | Cell-cell adhesion; Regulation of transcription, DNA-dependent; RNA splicing; mRNA processing | DNA binding |
To be considered as changing, a phosphopeptide should have a ratio outside the range in which 95% of phosphopeptides ratios for the internal control replicates are found. Phosphopeptides shown are those where a confident assessment of a significant decrease or increase was found. To be included the data must have a -fold change <0.6 or > 1.5 with a p value of less than 0.05. The significant changes are shown in bold; the underlined values represent changes where the confidence levels were not met. Every spectrum has been manually checked to verify phosphorylation. The first three columns report the Accession Number (UniProtKB-SwissProt), the Gene and Protein name, and the primary sequence of the phosphopeptide identified, indicating in bold the aminoacid modified by phosphorylation and in brackets the phosphosite position in the full length protein. The next three columns report the log(2) of the iTRAQ ratios representing the changes in phosphorylation level occurring during the progression of the hematopoietic differentiation, comparing Bry+Flk1−:Bry−Flk1−, Bry+Flk1+:Bry+Flk1− and Bry+Flk1+:Bry−Flk1−. The last two columns show the Biological process and Molecular function classification for the reported phosphopeptide-derived proteins, according to the Gene Ontology annotations. Data from two independent biological replicate were merged into the table.