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Mendes"],"technology_type":["Mass Spectrometry","SWATH MS"],"software":["Not available"],"submitter_keywords":["Mesenchymal stem cell","proteomics","cell therapy","hypoxic ischemic encephalopathy","Mesenchymal stem cell; brain hypoxia; cell therapy; hypoxic ischemic encephalopathy; preconditioning; proteomics","brain hypoxia","preconditioning"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD060639"],"tissue":["Brain"],"sample_protocol":["For each experimental condition, eight samples were collected, each corresponding to an individual animal, except the control group which had six samples. Upon thawing, the cerebellum and brainstem were removed from each sample. Tissue samples were homogenized in 2× Laemmli Sample Buffer at a ratio of 2.5 ml buffer per 600 mg of tissue. Samples were sonicated twice for 40 seconds each (3 seconds ON, 2 seconds OFF) at 60% amplitude. The homogenates were boiled at 95°C for 10 minutes with agitation at 300 rpm. After denaturation, samples were alkylated with 40% acrylamide (Biorad). The samples were centrifuged at 15,000 × g for 15 minutes. Protein concentration was determined using the Pierce™ 660nm Protein Assay Kit (Thermo Fisher Scientific) with Ionic Detergent Compatibility Reagent (Thermo Fisher Scientific). Two micrograms of the recombinant protein Green Fluorescent Protein (GFP) was added to each sample as an internal standard. Protein samples were separated using a partially electrophoretic separation protocol as previously described – Short GeLC-MS. Protein samples (50 µg total protein of each sample for quantification) or pools of each experimental condition (70 µg total protein of each pool for identification) and electrophoresis was performed at 110 V for approximately 18 minutes. After electrophoresis, proteins were visualized with Colloidal Coomassie Blue (43). After staining, gels were and washed repeatedly with distilled water until the background was clear. Each lane was sliced into bands of equal size and were treated with 600 µL of destaining solution (50 mM ammonium bicarbonate and 30% acetonitrile). Samples were incubated under agitation at 700 rpm for 15 minutes and the process was repeated until the gel pieces appeared transparent.Then, gel pieces were dried in a SpeedVac concentrator at 60°C for 1 hour.  After dehydration, 0.01 mg/mL trypsin in 10 mM ammonium bicarbonate was added to each well and plate was incubated for 15 minutes. After this initial incubation, more 10 mM ammonium bicarbonate buffer was added to the wells and trypsin was let to react overnight at room temperature (approximately 16 hours). Following enzymatic digestion, peptides were extracted using a sequential extraction protocol with increasing acetonitrile concentrations. Three extraction solutions were prepared: Solution A (30% acetonitrile with 1% formic acid), Solution B (50% acetonitrile with 1% formic acid), and Solution C (98% acetonitrile with 1% formic acid). For SWATH/DIA-MS analysis, peptides from all gel sections were pooled for each sample, while for data-dependent acquisition mode (DDA), the gel sections were maintained separately. The combined extracts were dried in a SpeedVac concentrator at 60°C until complete dryness. Dried peptide samples were reconstituted in a solution containing 2% acetonitrile and 0.1% formic acid before analysis on NanoLC™ 425 System (Eksigent) coupled to a Triple TOF™ 6600 mass spectrometer (Sciex) with an electrospray ionization source (DuoSpray™ Ion Source from Sciex). All data were acquired using Analyst TF 1.8.1 software (Sciex).  Chromatographic separation of peptides was carried out using a Triart C18 Capillary Column 1/32” (12 nm, 3 μm, 150 mm × 0.3 mm, YMC, Dinslaken, Germany) and a Triart C18 Capillary Guard Column (0.5 mm × 5 mm, 3 μm, 12 nm, YMC) at 50 °C. The flow rate for the separation process was set at 5 µL/min. The mobile phases used for the gradient were mobile phase A (0.1% formic acid and 5% dimethyl sulfoxide in water) and mobile phase B (0.1% formic acid and 5% dimethyl sulfoxide in acetonitrile). The separation was performed with a gradient elution program, which involved a gradual increase in mobile phase B from 5% to 30% over the first fifty minutes, followed by a rapid ramp from 30% to 98% over two minutes (50-52 min), 98% over two minutes (52-54 minutes), 98% to 5% over two minutes (54-56 minutes), and equilibration phase during 10 minutes with mobile phase B with 5% acetonitrile. The ESI DuoSpray™ ionization source was operated in positive ion mode. Key instrument parameters included an ion spray voltage of 5500 V, and both the nebulizer gas (GS1) and curtain gas (CUR) were maintained at 25 psi. In DDA mode, full MS spectra were collected across a mass range of m/z 350-2250 with an accumulation time of 250 ms, followed by up to 100 MS/MS scans (m/z 100-1500) with a 30 ms accumulation time. The total cycle time was maintained at 3.3 s. Precursor ion selection was based on charge states between +1 and +5, with a minimum intensity threshold of 10 counts per second. In SWATH/DIA-MS mode, the mass spectrometer was operated in a looped product ion mode and specifically tuned to a set of 168 overlapping windows, covering the precursor mass range of 350-1250 m/z. A 50 ms survey scan (350-1250 m/z) is acquired at the beginning of each cycle, and SWATH-MS/MS spectra were collected from 100-1500 m/z for 20 ms resulting in a cycle time of 3.3 seconds."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Each band of pooled samples was acquired individually in DDA mode. The ion library of the precursor masses and fragment ions was generated in ProteinPilotTM (Sciex) by combining all files from the experimental group pools, and data was searched using the reviewed Rattus norvegicus Swiss-Prot database. An independent False Discovery Rate (FDR) analysis was used to assess the quality of identifications, with a target-decoy approach. PeakViewTM (Sciex) was used for the analysis of data obtained with SWATH/DIA-MS acquisition and to retrieve the relative protein quantification. First, the library generated with the DDA data was imported into the software, indicating the number of proteins obtained with a local FDR below 5%. Then, the SWATH/DIA-MS data files corresponding to each sample were loaded and processing was done with the unique peptides and an FDR below 1%. Data was normalized to the total intensity."],"omics_type":["Proteomics"],"labhead":["Graça Baltazar"],"instrument_platform":[""],"labhead_affiliation":["Faculty of Health Sciences, University of Beira Interior, Covilhã"],"submission_type":["PARTIAL"],"species":["Rattus Norvegicus (rat)"],"submitter_mail":["vera3m@gmail.com"],"publication":["41913036 Serrenho I, Mendes V, Caramelo I, Cardoso CMP, Manadas B, Baltazar G. Quieting the Storm: Hypoxia as a Strategy to Boost UC-MSC Therapies for Hypoxic-Ischemic Brain Lesions in Neonatal Rats. Stem Cell Rev Rep. 2026 22(4):1974-2000 10.1007/s12015-026-11089-6"],"submitter_affiliation":["Center for Neuroscience and Cell Biology, University of Coimbra"],"submitter_country":["Portugal"],"pubmed_title":["Quieting the Storm: Hypoxia as a Strategy to Boost UC-MSC Therapies for Hypoxic-Ischemic Brain Lesions in Neonatal Rats."],"pubmed_authors":["Serrenho Inês I, Mendes Vera V, Caramelo Inês I, Cardoso Carla M P CMP, Manadas Bruno B, Baltazar Graça G"],"additional_accession":[]},"is_claimable":false,"name":"Quieting the Storm: Hypoxia as a Strategy to Boost UC-MSC Therapies for Hypoxic-Ischemic Brain Lesions in Neonatal Rats","description":"Integrating stem cell therapies into clinical settings faces several challenges, particularly in achieving the high cell yields necessary for attaining therapeutic doses. Preconditioning with hypoxic conditions has shown promise in enhancing the mesenchymal stem cells (MSCs)’ reparative capabilities of the central nervous system. Recent evidence suggests that oxygen concentration and exposure duration can shape MSCs’ phenotypes, supporting the need for further optimization of this strategy in a way to achieve maximal repair.  Methods: Wistar Han rat pups from both sexes underwent hypoxic-ischemic (HIE) brain injury at postnatal day 10 using the Rice-Vannucci model and were treated with umbilical cord derived-MSCs (UC-MSCs) preconditioned with prolonged mild hypoxia (MH; 5% oxygen for 48 hours) or short severe hypoxia (SSH; 0.1% oxygen for 24 hours) two days later.  Results: Our results show that UC-MSCs’ were able to alleviate motor and cognitive deficits caused by the HI brain lesion. To investigate the molecular effects of hypoxia-preconditioned MSCs in the neonatal brain post-HIE, we employed untargeted proteomics on ipsilesional brain samples from control, HIE, HIE treated with naïve UC-MSCs, and HIE treated with SSH-preconditioned UC-MSCs groups, 30 days after lesion induction. This approach identified protein signatures related to injury and therapeutic intervention. Pathway enrichment analysis further revealed that administration of UC-MSCs preconditioned with short severe hypoxia significantly impacted neural signaling, protein synthesis, and energy metabolism pathways, pointing to long-term mechanisms that may support neuronal repair. Conclusion: These findings enhance our understanding of hypoxia-preconditioning in MSCs therapy in driving a positive therapeutic response, supporting the development of more effective and feasible treatments for neonatal hypoxic-ischemic brain injury.","dates":{"publication":"2026-04-27","submission":"2025-02-10"},"accession":"PXD060639","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","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:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:294671","NEWT:376619","NEWT:1748418","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:3760","NEWT:1584","NEWT:2086595","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:3357","NEWT:7719","NEWT:29073","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:2020713","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:2579791","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","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:3715","NEWT:2065263","NEWT:199225","NEWT:177437","NEWT:717966","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:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:10886","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:1955249","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:2714896","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:1608994","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:485","NEWT:56636","NEWT:2499525","NEWT:1122981","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:31345","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:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:235455","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:717989","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:1871049","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:30455","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:299467","NEWT:9541","NEWT:51350","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:542","NEWT:2903","NEWT:185579","NEWT:872227","NEWT:13076","NEWT:2587410","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:2126337","NEWT:550","NEWT:253237","NEWT:10804","NEWT:33941","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","N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