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Nonnis"],"technology_type":["Mass Spectrometry","Top-down proteomics"],"software":["Not available"],"submitter_keywords":["Nanolc-ms/ms","Proteomics"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD059591"],"tissue":["Cell Suspension Culture"],"sample_protocol":["To analyse the secretome and transcriptome changes induced by collagen I mutations and by treatment with the chemical chaperone 4-PBA, three biological replicates were collected from primary osteoblasts isolated from two murine models of OI, Amish and Brtl mice (see Calvarial osteoblasts culture and treatment). This setup results in eight conditions (Col1a1+/+, Col1a1+/+ 4-PBA, Col1a1+/G349C, Col1a1+/G349C 4-PBA, Col1a2+/+, Col1a2+/+ 4-PBA, Col1a2+/G610C, Col1a2+/G610C 4-PBA), comprising twenty-four samples. Following 4-PBA treatment, the protein expression profile and quantitation was evaluated by LC-ESI-MS/MS, while gene expression was analysed by array and qPCR. OBs isolated from wild type (WT) littermates were designated as the control condition.  OBs were plated in two 10 cm petri dish for each condition and let grow until confluence. After three washes in α-MEM without FBS, the medium was changed with α-MEM without FBS, 50 μg/mL sodium ascorbate, 4 mM glutamine, 100 µg/mL penicillin and streptomycin with and without 5 mM 4-PBA and cells were incubated for 16 h. Conditioned medium was harvested and centrifuged at 2000 g for 10 min at 4°C. Proteins were quantified by quantum protein bicinchoninic protein assay kit (Euroclone). Bovine serum albumin (BSA) (Sigma-Aldrich) was used as standard. At least 1 mg of total protein was obtained for each sample. Liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analysis  Proteins were precipitated with 10% tricholoracetic acid for 2 h on ice, reduced, carbamidomethylated and digested with trypsin for 16 h at 37 °C using a protein trypsin ratio of 20:1. Nano LC-ESI-MS/MS analysis was performed on a Dionex UltiMate 3000 HPLC System directly connected to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) by a nanoelectrospray ion source. Peptide mixtures were enriched on 75 μm ID × 150 mm EASY-Spray PepMap RSLC C18 column (Thermo Fisher Scientific) and separated using the LC gradient: 1% acetonitrile (ACN) in 0.1% formic acid for 10 min, 1–4% ACN in 0.1% formic acid for 6 min, 4–30% ACN in 0.1% formic acid for 147 min and 30–50% ACN in 0.1% formic for 3 min at a flow rate of 0.3 μL/min. Orbitrap MS spectra were collected over an m/z range of 375–1500 at resolution of 120,000, operating in a data-dependent mode with a cycle time of 3 s between master scans. HCD MS/MS spectra were acquired in Orbitrap at resolution of 15,000 using a normalized collision energy of 35%, and an isolation window of 1.6 m/z. Dynamic exclusion was set to 60 s. Rejection of +1, and unassigned charge states were enabled. Data acquisition was controlled by Xcalibur 2.0 and Tune 2.4 software. Mass spectra were analyzed using MaxQuant software (version 1.6.1.0). The spectra were searched by the Andromeda search engine against the murine UniProt sequence database (released 22 September 2021). Protein identification required at least one unique or razor peptide per protein group. The initial maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.5 Da for MS/MS peaks. Enzyme specificity was set to trypsin, defined as C-terminal to Arg and Lys excluding Pro, and a maximum of two missed cleavages were allowed. Carbamidomethylcysteine was set as a fixed modification, while N-terminal acetylation, Met oxidation and Asn/Gln deamidation were set as variable modifications. Quantification in MaxQuant was performed using the built in XIC-based label free quantification (LFQ) algorithm using fast LFQ. The required false positive rate was set to 1% at the peptide and 1% at the protein level, and the minimum required peptide length was set to 7 amino acids."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Biological triplicates for each experiment were performed. Quantitative variables were expressed as mean ± standard deviation (SD). For the proteomic data, statistical analyses were performed using the Perseus software (version 1.45.05.63). For the proteomic analyses, Oonly proteins present and quantified in 3 out of 3 biological repeats were considered and used for statistical analysis. One-way ANOVA was applied to evaluate genotype and treatment effect. A p value ≤ < 0.05 was considered significant."],"omics_type":["Proteomics"],"labhead":["Elisa Maffioli"],"instrument_platform":[""],"labhead_affiliation":["DIVAS, University of Milan"],"submission_type":["PARTIAL"],"species":["Mus Musculus (mouse)"],"submitter_mail":["simona.nonnis@unimi.it"],"publication":["41937492 Besio R, Maffioli E, Palladino E, Sala A, Garibaldi N, Izzi V, Forlino A, Tedeschi G. OMICS Profiling Identifies Signatures of Senescence in Osteogenesis Imperfecta Osteoblasts Counteracted by 4-PBA. J Cell Mol Med. 2026 30(7):e71120 10.1111/jcmm.71120"],"submitter_affiliation":["University of Milan"],"submitter_country":["Italy"],"pubmed_abstract":["Mutations in collagen I are the most common cause of osteogenesis imperfecta (OI), leading to delayed protein folding and structurally abnormal molecules. While some aberrant collagen is secreted into the extracellular matrix (ECM), impairing bone quality, a significant fraction is retained intracellularly, disrupting osteoblast homeostasis. 4-phenylbutyrate (4-PBA) has been shown to improve osteoblast function and ECM composition in OI models. To investigate the intracellular consequences of mutant collagen retention and the mechanisms of 4-PBA, we analysed the secretome and transcriptome of two dominant OI mouse models, Col1a1<sup>+/G349C</sup> and Col1a2<sup>+/G610C</sup>. MS/MS proteomic analysis of conditioned media revealed senescence-associated secretory phenotype proteins, together with components linked to altered cytoskeletal organization and cell adhesion. Transcriptomic analysis identified P53 as a central hub gene, supporting premature senescence activation. Increased senescence-associated β-galactosidase activity, elevated expression of the cyclin-dependent kinase inhibitor P16, and reduced Ki67 levels further supported a senescent phenotype. Notably, senescence-associated proteins were absent from the secretome following 4-PBA treatment, which also modulated cytoskeletal and adhesion-related protein expression. Moreover, 4-PBA significantly reduced senescence marker expression and decreased the number of senescent cells. Overall, these findings indicate that cellular senescence underlies osteoblast dysfunction in OI and uncover a novel contribution of 4-PBA to osteoblast homeostasis."],"pubmed_title":["OMICS Profiling Identifies Signatures of Senescence in Osteogenesis Imperfecta Osteoblasts Counteracted by 4-PBA."],"pubmed_authors":["Besio Roberta R, Maffioli Elisa E, Palladino Erika E, Sala Alessandra A, Garibaldi Nadia N, Izzi Valerio V, Forlino Antonella A, Tedeschi Gabriella G"],"additional_accession":[]},"is_claimable":false,"name":"OMIC analyses identified senescence activation in osteogenesis imperfecta osteoblasts that is rescued by 4-PBA treatment","description":"Mutations in collagen I account for the most frequent cause of the skeletal disease osteogenesis imperfecta (OI) and are responsible for delay in protein folding and synthesis of molecules with abnormal structure. A fraction of aberrant collagen molecules is secreted in the extracellular matrix (ECM) where they impair the bone quality, while others are intracellularly retained in the endoplasmic reticulum where they perturb osteoblast homeostasis. The chemical chaperone 4-phenylbutyrate (4-PBA) improves OI osteoblast activity ameliorating ECM composition. To evaluate the consequences of intracellular malfunctioning in OI and to elucidate the mechanism responsible for the described positive effect of 4-PBA in OI, omics techniques were applied to analyze osteoblasts secretome and transcriptome in two well characterized murine models of dominant OI, the Col1a1+/G349C and the Col1a2+/G610C mice. Analyses of the cell culture media by nano LC-ESI-MS/MS and bioinformatic tools identified several senescence associated secretory proteins together with changes in cytoskeletal and cell adhesion proteins.  Bioinformatic analyses of transcriptomic data revealed p53 as hub gene supporting a premature senescence activation in both models. Increased senescence-associated β-galactosidase activity, as well as increased gene expression of the cyclin-dependent kinase-inhibitor p16, and a decreased expression of Ki67, indicating a permanent exit from the cell cycle in both mutants confirmed the ainduction of cellular senescence. Senescence proteins were undetectable in secretome collected following 4-PBA incubation that also modified the cytoskeletal and adhesion protein expression. Furthermore, the drug normalized the senescence markers expression and the number of senescent cells. Our study identified senescence as OI osteoblasts hallmark and discover a new positive contribution of 4-PBA to osteoblasts homeostasis.","dates":{"publication":"2026-04-13","submission":"2025-01-09"},"accession":"PXD059591","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:2042546","NEWT:259447","NEWT:295546","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:2065263","NEWT:177437","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:485","NEWT:56636","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:906914","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:550","NEWT:33941","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:9995","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:1613","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:298176","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:40674","NEWT:1194669","NEWT:74426","NEWT:443906","NEWT:13037","NEWT:519","NEWT:2510939","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:105487","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:300641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