Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063533/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063533/XA04879PISTR1_R1_Fr2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063533/XA04879PISTR1_R1_Fr1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD063533/XA04879PISTR1.zipprimaryOK200maqian57@163.comQian MaGlycoproteomicsMass SpectrometryDisease FreeNot availablehttps://www.ebi.ac.uk/pride/archive/projects/PXD063533Cell CultureEmbryonic Stem CellProtein Extraction The harvested ESCs pellet was transferred to a 5mL centrifuge tube and resuspended in four volumes of ice-cold lysis buffer (8 M urea, 1% protease inhibitor cocktail, 1% phosphatase inhibitor). The suspension was then subjected to ultrasonication for 3 minutes. After centrifugation at 12,000 g for 10 minutes at 4C, the cellular debris was effectively removed. The resulting supernatant was carefully collected, and the protein concentration was quantified using a BCA assay kit according to the manufacturer instructions. A total of 0.5 mg of protein was prepared for subsequent experimental procedures. Trypsin Digestion For protein digestion, the protein solution was first reduced with 5 mM dithiothreitol at 56C for 30 minutes, followed by alkylation with 11 mM iodoacetamide at room temperature in darkness for 15 minutes. The sample was then diluted with 200 mM TEAB to reduce the urea concentration to less than 2 M. Trypsin was added at a 1:50 trypsin-to-protein mass ratio for the initial overnight digestion, followed by a second digestion at a 1:100 trypsin-to-protein mass ratio for 4 hours. The resulting peptides were desalted using a Strata X SPE column. Modification Enrichment Peptide mixtures were first incubated with a suspension of IMAC microspheres in loading buffer (50% acetonitrile/0.5% acetic acid). To remove non-specifically bound peptides, the IMAC microspheres were sequentially washed with 50% acetonitrile/0.5% acetic acid and 30% acetonitrile/0.1% trifluoroacetic acid. Enriched phosphopeptides were then eluted by adding an elution buffer containing 10% NH4OH, and the elution was performed under agitation. The supernatant containing the phosphopeptides was collected and lyophilized prior to LC-MS/MS analysis.PrideDatabase Search The resulting MS/MS data were processed using PD search engine (v.2.4). Tandem mass spectra were searched against Mus_musculus_10090_SP_20230103.fasta (17132 entries) concatenated with reverse decoy database. Trypsin (full) was specified as cleavage enzyme allowing up to 2 missing cleavages. The minimum peptide length was set to 6 amino acid residues. The maximum number of peptide modifications was set to 3. The mass error tolerance of primary parent ion is set to 10 ppm, and the mass error tolerance of secondary fragment ion is set to 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification, and Oxidation (M), Acetyl (N-terminus), Met-loss (M), Met-loss acetyl (M), Phospho (S, T, Y) were specified as variable modifications. The false discovery rate (FDR) was adjusted to < 1%.ProteomicsQian MaPARTIALPeking University First HospitalMus Musculus (mouse)10.1002/PMIC.70147maqian57@163.comPeking University First HospitalChinafalseHigh-throughput phosphoproteomic of mESCsPhosphorylation is a key post-translational modification involved in many cellular processes. Embryonic stem cells (ESCs), characterized by their self-renewal capacity and pluripotent differentiation potential, are widely used in studies of developmental biology and regenerative medicine. However, existing phosphoproteomic data for ESCs remain limited in throughput, restricting our understanding of phosphorylation-mediated regulatory mechanisms. In this study, we performed high-throughput phosphoproteomic profiling and identified 3,711 phosphoproteins and 11,410 phosphosites.2026-05-222025-05-01PXD063533NEWT:6945NEWT:3555NEWT:241368NEWT:2NEWT:157546NEWT:190802NEWT:35554NEWT:150475NEWT:9417NEWT:347515NEWT:1216979NEWT:307972NEWT:544496NEWT:5180NEWT:256737NEWT:115104NEWT:1081927NEWT:67825NEWT:13076NEWT:1249668NEWT:376741NEWT:317NEWT:1736309NEWT:7227NEWT:7469NEWT:885318NEWT:876138NEWT:4081NEWT:554NEWT:98334NEWT:237561NEWT:10036NEWT:7574NEWT:1351NEWT:7215NEWT:272563NEWT:507601NCBITaxon:79857NCBITaxon:6157NEWT:95648NEWT:746360NEWT:6239NEWT:1589NEWT:470150NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:4054NEWT:3880NEWT:8782NEWT:1000589NEWT:1902NEWT:85962NEWT:160488NEWT:28104NEWT:317447NEWT:7955NCBITaxon:2NEWT:985076NEWT:7959NEWT:2261NEWT:4565NEWT:1264690NEWT:6192NEWT:28532NCBITaxon:38727NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:216595NEWT:243230NEWT:8355NEWT:931281NEWT:7029NEWT:1283300NEWT:334747NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:3218NEWT:5759NEWT:1736231NEWT:436486NEWT:6287NEWT:2242NEWT:300641NEWT:4784NEWT:727NEWT:9796NEWT:725NEWT:360106NEWT:260707NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:83334NEWT:83332NEWT:29760NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:1148NEWT:11676NEWT:55571NEWT:100226NCBITaxon:6073NEWT:4530NEWT:4896NEWT:6279NEWT:7370NEWT:6282NEWT:1134506NEWT:575584NEWT:1773NEWT:38783NEWT:8727NEWT:1895NEWT:1182590NEWT:8726NEWT:10090NEWT:935293NEWT:749200NEWT:4120NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:990346NEWT:5334NEWT:145953NEWT:257309NEWT:230741NEWT:284812NCBITaxon:10359NCBITaxon:1313NEWT:43330NEWT:242619NEWT:44544NEWT:373995NEWT:544404NEWT:3702NEWT:129249NEWT:8839NEWT:4232NEWT:990119NEWT:4113NEWT:11298NEWT:171101NEWT:196627NEWT:408172NEWT:5691NEWT:408170NEWT:493760NEWT:260710NEWT:627025NEWT:400772NEWT:1097677NEWT:3708NEWT:106592NEWT:9913NEWT:1432138NEWT:10312NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:8030NEWT:29722NEWT:380394NEWT:1692259NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:135588NEWT:1843183NEWT:95486NEWT:58002NEWT:9103NEWT:4577NEWT:5664NEWT:2157NEWT:146479NEWT:10306NEWT:1911079NEWT:145943NEWT:3635NEWT:5811NEWT:235443NEWT:1480154NEWT:1274414NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:884019NEWT:169963NEWT:36329NEWT:9606NEWT:367830NEWT:157295NEWT:410289NEWT:373153NEWT:915099NEWT:74940NEWT:1450511NEWT:470NEWT:84023NEWT:9838NCBITaxon:9615NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:2762NEWT:5476NEWT:1174673NEWT:562NEWT:1274432NEWT:1274426NEWT:1423NEWT:4932NEWT:70448NEWT:9825NEWT:1274423NEWT:3603NEWT:698936NEWT:2759NEWT:3847NEWT:39946NEWT:9823NEWT:9940NEWT:573NEWT:9031NEWT:1274420NEWT:7091