Molecule transition lists, including compound names, expected retention times, parent ions, all fragment ions and their respective collision energies were uploaded to the software.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - positive - hilic"],"chromatography_protocol":["LC-MS/MS analysis was performed on a Waters Premier Acquity UPLC. Samples were randomized prior to data acquisition. Analytes were separated on a Waters Acquity Premier BEH Amide 1.7 µm VanGuard FIT 2.1 x 150 mm HILIC column, with a guard column, held at 40 °C, using the following mobile phases at 0.25 mL/min: A:10 mM ammonium acetate in H2O, w. 5 µM medronic acid; B: 10 mM ammonium acetate in 90% ACN, w. 5 µM medronic acid. TThe total run time was 7.5 min and the gradient for separation was as follows : 0 min, 10% A; 0.5 min, 10%A ; 1 min, 35% A; 3.5 min, 41.5% A; 4.5 min, 43% A; 6 min, 55% A; 6.5-7.5 min, 10%A
"],"publication":["Multi-omics Characterization of Acquired Olaparib Resistance in BRCA1 and BRCA2 Mutant Breast Cancer Cell Lines. 10.1016/j.mcpro.2025.101034. PMID:40669753"],"submitter_affiliation":["University of Copenhagen"],"submitter_name":["Matthias Anagho-Mattanovich"],"organism_part":["epithelial cell line","breast cancer cell line","mixture","solvent"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["The tubes were repeatedly submerged in liquid nitrogen to snap freeze and then vortexed. After the third repetition, 50 µL of 15% ammonium carbonate was added to neutralize the mix. Samples were left on ice for 1 h, then centrifuged at 13,000 rpm for 15 minutes at 4 °C, then stored at -80 °C until analysis. Prior to analysis, samples were randomized, thawed and vortexed, then 50 µL was aliquoted into LC vials with inserts. Quality control pools for each type were created by combining 20 µL from each sample (parent or clone) and then aliquoted. Extraction blanks and solvent blanks were also run, as quality control samples. These samples were run together with the first calibration curve.
Calibration standards were prepared using a mix of adenosine diphosphate ribose (ADPR), NAD, NADH+, and Nam in methanol, with concentrations determined based on the measured levels in pooled samples. Internal standards (10 μg/ml NA-d4, NAD-d4, NADH-d4, Nam-d4) were added to all calibration points and extracted similarly to the cell samples. A second calibration curve for nicotinic acid (NA), nicotinamide riboside (NR), nicotinic acid adenine dinucleotide (NAAD), nicotinamide mononucleotide (NMN), and nicotinic acid mononucleotide (NAMN) was prepared and up-concentrated post-extraction by drying and resuspension in a reduced volume.
"],"organism":["blank","reference compound","Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS12109"],"author":["Michael Nielsen. Evosep Biosystems. Billedskærervej 15, 5230 Odense. mln@evosep.com."],"data_transformation_protocol":["Data was processed using Skyline (v. 24.1.0.199). Molecule transition lists, including compound names, expected retention times, parent ions, all fragment ions and their respective collision energies were uploaded to the software. Samples, blanks, QCs and calibration points were loaded, peaks picked automatically and integrated from baseline to baseline. Calibration curves were plotted (weighted 1/x2) using the average ratio between each compound peak area to the peak area for the corresponding labelled internal standard, if available, or NAD-d4 (for NADH and ADPR) and NA-d4 (for NR). Any calibration points with a calculated concentration accuracy of more or less than 15% were discarded (marked in grey in the sample table). Pool samples were used as quality control, with relative standard deviation values under 25%. The limit of quantitation (LoQ) was defined as the lowest concentration that could be reliably quantified, i.e. where peak height = 10x the S/N ratio. The concentration of NMN, NAMN, NA and NAAD were found to be below the LoQ in all samples (below LoD for NMN).
"],"study_factor":["Cell line"],"submitter_email":["matthias.mattanovich@sund.ku.dk"],"sample_collection_protocol":["Immortalized breast cancer cell lines MDAMB436 and HCC1428, originally from the American Type Culture Collection, were kindly provided by Jesper Olsen (NNF-CPR, University of Copenhagen). Cells were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/ml; Gibco) (cell culture medium) in a 37 °C incubator with 5% CO2.
Media from the cells were collected (approx. 500 µL) and stored at -80 °C. The adherent cells were rinsed with 1 mL/well of phosphate buffered saline (PBS). The PBS was carefully removed and 360 µL of 90% methanol containing 5 µg/mL of internal standard mix was added (NA-d4, NAD-d4, NADH-d4 and Nam-d4), to quench the cells. Adherent cells were scraped, the whole mixture was transferred to a microcentrifuge tube and 90 µL of 100 mM formic acid was added.
"],"omics_type":["Metabolomics"],"study_design":["Multi-omics study","breast cancer","BRCA1","BRCA2","Drug resistance","targeted metabolites"],"curator_keywords":["Multi-omics study","breast cancer","BRCA1","BRCA2","Drug resistance","targeted metabolites"],"mass_spectrometry_protocol":["The mass spectrometer, Waters Xevo TQ-XS triple quadrupole mass spectrometer, was equipped with an electrospray ionization source and operated in positive mode, at a capillary voltage of 0.6 V, and a cone voltage of 15 V. The desolvation temperature was 600 °C, gas flow was 1000 L/h and cone gas flow 150 L/h. T
The MRM settings for the method are follows: Compound: Retention time (min); Parent ion (m/z); Fragment 1 (m/z); Fragment 1 CE (eV); Fragment 2 ion (m/z); Fragment 2 CE (eV): Nam: 2.15; 122.9; 96; 14:79.9; 18/ Nam-d4; 2.15;127; 83;18; 84; 18/ NA: 2.95; 123.9; 106;14; 77.6; 18/ NA-d4: 2.95; 128; 81; 10;81; 10/ NR: 3.87; 254.9; 106; 30; 122.9; 10/ NADH: 3.47; 666; 514; 25; 136; 40/ NADH-d4: 3.47; 670; 112; 43; 136; 43/ ADP: 3.53;559.9;347;14;135.9;32/ NAD: 3.66; 664; 428;30; 136; 30/ NAD-d4: 3.66; 668; 136; 43/ NAAD: 3.57; 665.05; 428; 26; 135.9; 44/ NAMN: 3.68; 336.01; 524; 18; 96.8; 26/ NMN: 4.62; 334.96; 97; 26; 122.9; 12.
"],"metabolite_name":["Nicotinamide Riboside","NADH","Nadide","Nicotinic acid","deamido-NAD","Nicotinamide","Nicotinate D-ribonucleotide","Adenosine diphosphate ribose","nmn"],"pubmed_abstract":["Poly (ADP-ribose) polymerase inhibitors (PARPi) are widely used as targeted therapies against breast cancers with BRCA mutations. However, the development of resistance to PARPi poses a significant challenge for long-term efficacy of these therapies, warranting further understanding of mechanisms of PARPi resistance. Here, we generated and characterized Olaparib resistance in BRCA1/2 mutant breast cancer cell lines MDAMB436 and HCC1428 using a systems-level multi-omics approach, including transcriptome, proteome, phosphoproteome, and ADP-ribosylation analysis. Our analyses revealed that resistance development strongly correlated with protein expression changes, while modest effects on phosphorylation- and ADP-ribosylation-dependent signaling pathways were observed. We found that BRCA1 expression was reestablished in OR MDAMB436 cell lines, while PARP1 expression was decreased. In OR HCC1428 cell lines, the BRCA2 mutation was not reverted. However, we observed increased expression of Fanconi anemia group D2 (FANCD2), histone parylation factor 1 (HPF1), and Nicotinamide phosphoribosyltransferase (NAMPT) in various cell lines, suggesting increased replication fork protection, and changes in the ADPr pathway and adaptation of metabolic pathways as resistance mechanisms. Our findings provide valuable insights into the complex landscape of PARPi resistance, offering potential targets for further investigation and therapeutic intervention."],"pubmed_title":["Multi-omics Characterization of Acquired Olaparib Resistance in BRCA1 and BRCA2 Mutant Breast Cancer Cell Lines."],"pubmed_authors":["Anagho-Mattanovich Holda A HA, Mullari Meeli M, Anagho-Mattanovich Matthias M, Cho Hayoung H, Pedersen Anna-Kathrine AK, Palasca Oana O, Olsen Jesper V JV, Locard-Paulet Marie M, Nielsen Michael L ML"],"additional_accession":[]},"is_claimable":false,"name":"Multi-omics characterization of acquired Olaparib resistance in BRCA1 and BRCA2 mutant breast cancer cell lines","description":"Poly (ADP-ribose) polymerase inhibitors (PARPi) are widely used as targeted therapies against breast cancers with BRCA mutations. However, the development of resistance to PARPi poses a significant challenge for long-term efficacy of these therapies, warranting further understanding of mechanisms of PARPi resistance. Here, we generated and characterized Olaparib resistance (OR) in BRCA1/2 mutant breast cancer cell lines MDAMB436 and HCC1428 using a systems-level multi-omics approach, including transcriptome, proteome, phosphoproteome, and ADP-ribosylation analysis. Our analyses revealed that resistance development strongly correlated with protein expression changes, while modest effects on phosphorylation- and ADP-ribosylation-dependent signaling pathways were observed. We found that BRCA1 expression was reestablished in OR MDAMB436 cell lines, while PARP1 expression was decreased. In OR HCC1428 cell lines, the BRCA2 mutation was not reverted. However, we observed increased expression of Fanconi anemia group D2 (FANCD2), histone parylation factor 1 (HPF1, and Nicotinamide phosphoribosyltransferase (NAMPT) in various cell lines, suggesting increased replication fork protection, and changes in the ADPr pathway and adaptation of metabolic pathways as resistance mechanisms. Our findings provide valuable insights into the complex landscape of PARPi resistance, offering potential targets for further investigation and therapeutic intervention.","dates":{"publication":"2026-05-11","submission":"2025-03-09"},"accession":"MTBLS12109","cross_references":{"MetaboLights":["MTBLC17154","MTBLC15940","MTBLC15927","MTBLC16908","MTBLC16960","MTBLC15846","MTBLC18304","MTBLC15763","MTBLC14649"],"pubmed":["40669753"],"ChEBI":["CHEBI:17154","CHEBI:15940","CHEBI:15927","CHEBI:16908","CHEBI:16960","CHEBI:15846","CHEBI:18304","CHEBI:15763","CHEBI:14649"]}}