#METABOLOMICS WORKBENCH AhmetUcar_20230622_061742 DATATRACK_ID:4108 STUDY_ID:ST002753 ANALYSIS_ID:AN004467 PROJECT_ID:PR001714 VERSION 1 CREATED_ON June 27, 2023, 12:55 pm #PROJECT PR:PROJECT_TITLE Metabolomics of intact murine mammary ducts at a pre-cancerous stage PR:PROJECT_TYPE LC/MS PR:PROJECT_SUMMARY Metabolomics analyses of intact mammary ducts isolated from 8-week-old PR:PROJECT_SUMMARY MMTV-Neu-IRES-Cre mice and their wildtype littermates PR:INSTITUTE University of Manchester PR:LAST_NAME Ucar PR:FIRST_NAME Ahmet PR:ADDRESS Oxford Road, Manchester, Lancashire, M13 9PT, United Kingdom PR:EMAIL ahmet.ucar@manchester.ac.uk PR:PHONE 00447928839333 PR:FUNDING_SOURCE Breast Cancer Now (BCN) #STUDY ST:STUDY_TITLE Comparative analyses of metabolome for the intact mammary ducts of 8-week-old ST:STUDY_TITLE MMTV-Neu-IRES-Cre mice versus their wildtype littermates ST:STUDY_TYPE Comparative ST:STUDY_SUMMARY Transgenic overexpression of HER2/Neu oncogene in murine mammary glands result ST:STUDY_SUMMARY in mammary tumor formation. To elucidate the potential changes in the metabolic ST:STUDY_SUMMARY activities of HER2/Neu-overexpressing mammary epithelia prior to any obvious ST:STUDY_SUMMARY tumorigenic growth, we isolated intact mammary ducts from the No:4 inguinal ST:STUDY_SUMMARY mammary glands of 8-week-old MMTV-Neu-IRES-Cre and WT littermate mice via mild ST:STUDY_SUMMARY collagenase treatment and performed a LC/MS based untargeted metabolomics assay. ST:INSTITUTE Manchester Institute of Biotechnology, University of Manchester ST:LAST_NAME Ucar ST:FIRST_NAME Ahmet ST:ADDRESS Michael Smith building, Oxford Road, Manchester, M13 9PT, UK ST:EMAIL ahmet.ucar@manchester.ac.uk ST:PHONE 00447928839333 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 8-week-old SU:GENDER Female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 407 660 Genotype:Wildtype RAW_FILE_NAME=407 660.mzML SUBJECT_SAMPLE_FACTORS - 407 661 Genotype:Wildtype RAW_FILE_NAME=407 661.mzML SUBJECT_SAMPLE_FACTORS - 407 662 Genotype:MMTV-NIC RAW_FILE_NAME=407 662.mzML SUBJECT_SAMPLE_FACTORS - 407 663 Genotype:MMTV-NIC RAW_FILE_NAME=407 663.mzML SUBJECT_SAMPLE_FACTORS - 407 667 Genotype:MMTV-NIC RAW_FILE_NAME=407 667.mzML SUBJECT_SAMPLE_FACTORS - 407 668 Genotype:MMTV-NIC RAW_FILE_NAME=407 668.mzML SUBJECT_SAMPLE_FACTORS - 407 669 Genotype:Wildtype RAW_FILE_NAME=407 669.mzML SUBJECT_SAMPLE_FACTORS - QC1 Genotype:Quality Control RAW_FILE_NAME=QC1.mzML SUBJECT_SAMPLE_FACTORS - QC2 Genotype:Quality Control RAW_FILE_NAME=QC2.mzML SUBJECT_SAMPLE_FACTORS - QC3 Genotype:Quality Control RAW_FILE_NAME=QC3.mzML SUBJECT_SAMPLE_FACTORS - QC4 Genotype:Quality Control RAW_FILE_NAME=QC4.mzML SUBJECT_SAMPLE_FACTORS - QC5 Genotype:Quality Control RAW_FILE_NAME=QC5.mzML SUBJECT_SAMPLE_FACTORS - QC6 Genotype:Quality Control RAW_FILE_NAME=QC6.mzML SUBJECT_SAMPLE_FACTORS - Blank Genotype:Blank RAW_FILE_NAME=blank01.mzML #COLLECTION CO:COLLECTION_SUMMARY Intact mammary ducts were isolated from No:4 glands of 8-week-old mice using a CO:COLLECTION_SUMMARY mild collagenase treatment and size selection using a 100 um mesh. CO:SAMPLE_TYPE Breast #TREATMENT TR:TREATMENT_SUMMARY There was no additional treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Ductal organoids were centrifuged, tissue pellets were snap-frozen in liquid SP:SAMPLEPREP_SUMMARY nitrogen and then resuspended in 50% Methanol to incubate overnight at 4 °C. SP:SAMPLEPREP_SUMMARY Next day, samples were centrifuged at 20,000 x g for 3 min and the top 50 µl SP:SAMPLEPREP_SUMMARY supernatant was transferred to a glass autosampler vial with 300 µl insert and SP:SAMPLEPREP_SUMMARY capped. Quality control samples were made by pooling 10 µl from each sample. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY A sample volume of 5 μL was injected by pulled loop onto a 5 μL sample loop CH:CHROMATOGRAPHY_SUMMARY with 150 μl post-injection needle wash with 9:1 acetonitrile and water. CH:CHROMATOGRAPHY_SUMMARY Injection cycle time was 1 min per sample. Separations were performed using an CH:CHROMATOGRAPHY_SUMMARY Agilent Poroshell 120 HILIC-Z column with dimensions of 150 mm length, 2.1 mm CH:CHROMATOGRAPHY_SUMMARY diameter and 2.7 μm particle size equipped with a guard column of the same CH:CHROMATOGRAPHY_SUMMARY phase. Mobile phase A was water with 10 mM ammonium acetate adjusted to pH 9 CH:CHROMATOGRAPHY_SUMMARY with ammonium hydroxide and 20 µM medronic acid, mobile phase B was 85:15 CH:CHROMATOGRAPHY_SUMMARY acetonitrile and water with 10 mM ammonium acetate adjusted to pH 9 with CH:CHROMATOGRAPHY_SUMMARY ammonium hydroxide and 20 µM medronic acid. Separation was performed by CH:CHROMATOGRAPHY_SUMMARY gradient chromatography at a flow rate of 0.25 ml/min, starting at 96 % B for 2 CH:CHROMATOGRAPHY_SUMMARY minutes, ramping to 65 % B over 20 min, hold at 65 % B for 2 min, then back to CH:CHROMATOGRAPHY_SUMMARY 96 % B. Re-equilibration time was 5 min. Total run time including 1 min CH:CHROMATOGRAPHY_SUMMARY injection cycle was 30 min. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Agilent Poroshell 120 HILIC-Z column (150 mm length, 2.1 mm diameter and 2.7 μm CH:COLUMN_NAME particle size) CH:SOLVENT_A 100% water; 10 mM ammonium acetate (pH 9); 20 µM medronic acid CH:SOLVENT_B 85% acetonitrile/ 15% water; 10 mM ammonium acetate (pH 9); 20 µM medronic acid CH:FLOW_GRADIENT starting at 96 % B for 2 minutes, ramping to 65 % B over 20 min, hold at 65 % B CH:FLOW_GRADIENT for 2 min, then back to 96 % B CH:FLOW_RATE 0.25 ml/min CH:COLUMN_TEMPERATURE 50 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6600 TripleTOF MS:INSTRUMENT_TYPE Triple TOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The mass spectrometer ran in negative mode under the following source MS:MS_COMMENTS conditions: curtain gas pressure, 50 psi; ionspray voltage, -4500 V; MS:MS_COMMENTS temperature, 400 °C; ESI nebulizer gas pressure, 50 psi; heater gas pressure, MS:MS_COMMENTS -70 psi; declustering potential, -80 V. Data was acquired in an information MS:MS_COMMENTS dependent manner across 10 high sensitivity product ion scans, each with an MS:MS_COMMENTS accumulation time of 100 ms and a TOF survey scan with accumulation time of 250 MS:MS_COMMENTS ms. Total cycle time was 1.3 s. Collision energy was determined using the MS:MS_COMMENTS formula CE (V) = 0.084 x m/z +12 up to a maximum of 55 V. Isotopes within 4 Da MS:MS_COMMENTS were excluded from the scan. Acquired data were checked in PeakView 2.2 and MS:MS_COMMENTS imported into Progenesis Qi 2.4 for metabolomics, where they were aligned, peaks MS:MS_COMMENTS were picked, normalized to all compounds and deconvoluted according to standard MS:MS_COMMENTS Progenesis workflows. Annotations were made by searching the accurate mass, MS:MS_COMMENTS MS/MS spectrum and isotope distribution ratios of acquired data against the NIST MS:MS_COMMENTS MS/MS metabolite library. Metabolites were identified by searching retention MS:MS_COMMENTS times and accurate masses against an in-house chemical standard library. A MS:MS_COMMENTS validated identification is only given if identical hits are made against both MS:MS_COMMENTS the NIST MS/MS and in-house chemical standard libraries. Samples were grouped MS:MS_COMMENTS according to the mouse genotypes (i.e WT or MMTV-Neu) and were filtered based on MS:MS_COMMENTS the following statistical criteria – a maximum fold change between sample MS:MS_COMMENTS groups of at least 1.5-fold, ANOVA p values of <0.05, minimum CV of <30 % and a MS:MS_COMMENTS minimum normalized abundance of 10. MS:MS_RESULTS_FILE ST002753_AN004467_Results.txt UNITS:Normalized peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END