#METABOLOMICS WORKBENCH fernandezlab_20190416_195008 DATATRACK_ID:1706 STUDY_ID:ST001172 ANALYSIS_ID:AN001940 PROJECT_ID:PR000784 VERSION 1 CREATED_ON April 19, 2019, 1:13 pm #PROJECT PR:PROJECT_TITLE Deep Metabolomics of a High-Grade Serous Ovarian Cancer Triple Knockout Mouse PR:PROJECT_TITLE Model. PR:PROJECT_TYPE Untargeted metabolomics PR:PROJECT_SUMMARY High-grade serous carcinoma (HGSC) is the most common and deadliest ovarian PR:PROJECT_SUMMARY cancer (OC) type, accounting for 70–80% of OC deaths. This high mortality is PR:PROJECT_SUMMARY largely due to late diagnosis. Early detection is thus crucial to reduce PR:PROJECT_SUMMARY mortality. Yet tumor pathogenesis of HGSC remains poorly understood, making PR:PROJECT_SUMMARY early detection difficult. Faithfully and reliably representing the clinical PR:PROJECT_SUMMARY nature of human HGSC, a recently-developed triple knockout (TKO) mouse model PR:PROJECT_SUMMARY offers a unique opportunity to examine the entire disease spectrum of HGSC. Deep PR:PROJECT_SUMMARY metabolomics study was performed to serum samples collected from these mice to PR:PROJECT_SUMMARY understand the metabolic alternations associated with HGSC development and PR:PROJECT_SUMMARY progression, and provide guidance toward early detection. PR:INSTITUTE Georgia Institute of Technology PR:DEPARTMENT Chemistry PR:LABORATORY Fernández PR:LAST_NAME Huang PR:FIRST_NAME Danning PR:ADDRESS 901 Atlantic Dr NE, Atlanta, GA, 30332, USA PR:EMAIL dhuang74@gatech.edu PR:PHONE 404-512-7523 #STUDY ST:STUDY_TITLE Deep Metabolomics of a High-Grade Serous Ovarian Cancer Triple Knockout Mouse ST:STUDY_TITLE Model. ST:STUDY_TYPE Untargeted metabolomics ST:STUDY_SUMMARY Metabolic alternations were investigated by applying Ultra Performance Liquid ST:STUDY_SUMMARY Chromatography Mass Spectrometry (UPLC-MS) to serum samples collected from ST:STUDY_SUMMARY triple knockout (TKO) mice at pre-malignant, early, and advanced stages of HGSC. ST:STUDY_SUMMARY Samples were analyzed with control mice, which have the same genetic background ST:STUDY_SUMMARY as TKO mice but develop no tumors. To enhance the selectivity for HGSC-specific ST:STUDY_SUMMARY metabolite markers, a tumor control group was also included. These were uterine ST:STUDY_SUMMARY tumor (UT) mice that developed uterine tumors, but no HGSC. All samples were ST:STUDY_SUMMARY analyzed using reverse phase (RP) and hydrophilic interaction liquid ST:STUDY_SUMMARY chromatography (HILIC) UPLC-MS analysis in positive and negative ion modes. ST:INSTITUTE Georgia Institute of Technology ST:DEPARTMENT Chemistry ST:LABORATORY Fernández ST:LAST_NAME Huang ST:FIRST_NAME Danning ST:ADDRESS 901 Atlantic Dr NE ST:EMAIL dhuang74@gatech.edu ST:PHONE 4045127523 ST:NUM_GROUPS 5 ST:TOTAL_SUBJECTS 84 ST:NUM_FEMALES 84 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 2 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 3 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 4 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 5 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 6 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 7 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 8 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 9 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 10 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 11 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 12 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 13 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 14 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 15 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 16 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 17 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 18 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 19 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 20 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 21 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 22 Group:pre p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=cre/+ SUBJECT_SAMPLE_FACTORS - 23 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 24 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 25 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 26 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 27 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 28 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 29 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 30 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 31 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 32 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 33 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 34 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 35 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 36 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 37 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 38 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 39 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 40 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 41 Group:ctrl p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=+/+ SUBJECT_SAMPLE_FACTORS - 42 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 43 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 44 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 45 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 46 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 47 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 48 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 49 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 50 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 51 Group:ET p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 52 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 53 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 54 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 55 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 56 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 57 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 58 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 59 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 60 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 61 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 62 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 63 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 64 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 65 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 66 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 67 Group:LT p53=R172H/+; Pten=fl/fl; Dicer=fl/fl; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 68 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 69 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 70 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 71 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 72 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 73 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 74 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 75 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 76 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 77 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 78 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 79 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 80 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 81 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 82 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 83 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ SUBJECT_SAMPLE_FACTORS - 84 Group:UT p53=fl/fl; Pten=fl/fl; Dicer=+/+; Amhr2=Cre/+ #COLLECTION CO:COLLECTION_SUMMARY Blood samples were collected via retro-orbital bleeding after anesthesia, from CO:COLLECTION_SUMMARY 22 TKO-Pre mice (average age, 2.0 months; age range, 1.3–3.1), 10 TKO-ET mice CO:COLLECTION_SUMMARY (4.8 months: 2.5–5.9), 16 TKO-AT mice (6.4 months: 4.3–10.1), 19 TKO-ctrl CO:COLLECTION_SUMMARY mice (3.0 months: 3.0–8.2), and 17 UT mice (5.4 months: 3.7–6.8). Blood CO:COLLECTION_SUMMARY samples were centrifuged at 14,000 rpm for 5 minutes at room temperature, and CO:COLLECTION_SUMMARY serum was collected and stored at -80 °C until UPLC-MS analysis. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY Triple-mutant (TKO) mice (p53 LSL-R172H/+ Dicer1 flox/flox Pten flox/flox Amhr2 TR:TREATMENT_SUMMARY cre/+) were generated by mating p53 LSL-R172H/+ Dicer1 flox/flox Pten flox/flox TR:TREATMENT_SUMMARY female with Dicer1 flox/flox Pten flox/flox Amhr2 cre/+ male mice. Serum samples TR:TREATMENT_SUMMARY were collected from TKO mice at different stages of tumor progression: from TR:TREATMENT_SUMMARY premalignant stage to early stage to advanced stage. Precursor lesions are TR:TREATMENT_SUMMARY fallopian tubes at premalignant stage, when no tumors are yet present. TKO mice TR:TREATMENT_SUMMARY with precursor (i.e. premalignant stage) lesions in the fallopian tubes are TR:TREATMENT_SUMMARY referred to as “TKO-Pre”; TKO-ET (early tumor) are TKO mice with early-stage TR:TREATMENT_SUMMARY tumors in the fallopian tube without evidence of metastasis; TKO-AT TR:TREATMENT_SUMMARY (advanced-stage tumor) are TKO mice with ovarian and peritoneal metastases TR:TREATMENT_SUMMARY accompanied by ascites; and TKO-ctrl are control mice (p53 LSL-R172H/+ Dicer1 TR:TREATMENT_SUMMARY flox/flox Pten flox/flox), which have the same genetic background as TKO mice TR:TREATMENT_SUMMARY but develop no tumors. All TKO mice developed high-grade serous carcinoma (HGSC, TR:TREATMENT_SUMMARY i.e. high-grade serous ovarian cancer). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Serum samples were thawed on ice and subject to two different sample preparation SP:SAMPLEPREP_SUMMARY protocols to profile either non-polar or polar sub-metabolomes. Reverse phase SP:SAMPLEPREP_SUMMARY (RP) and hydrophilic interaction liquid chromatography (HILIC) UPLC-MS analysis SP:SAMPLEPREP_SUMMARY were used for these different polarity metabolite fractions to obtain SP:SAMPLEPREP_SUMMARY complementary information. Methanol (for polar) or iso-propanol (for non-polar) SP:SAMPLEPREP_SUMMARY was added to a 50 μL serum sample in a 3:1 ratio to precipitate proteins. SP:SAMPLEPREP_SUMMARY Samples were vortex mixed for 10 s and centrifuged at 13,000 rpm for 7 min. SP:SAMPLEPREP_SUMMARY Then, 150 μL supernatant was frozen at -80 °C for UPLC-MS analysis. A sample SP:SAMPLEPREP_SUMMARY blank was prepared with 50 μL of LC-MS grade water, and a pooled quality SP:SAMPLEPREP_SUMMARY control (QC) sample was created by mixing 10 μL aliquot of each serum sample. SP:SAMPLEPREP_SUMMARY Both the sample blank and the pooled sample were processed with the same SP:SAMPLEPREP_SUMMARY procedure as the murine serum samples. Solvent blanks and sample blanks were SP:SAMPLEPREP_SUMMARY analyzed together with murine serum samples. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters Acquity BEH HILIC (150 x 2.1mm, 1.7um) CH:COLUMN_TEMPERATURE 55 CH:SOLVENT_A water/acetonitrile (95:5 v/v), 10 mM ammonium acetate and 0.05 % ammonium CH:SOLVENT_A hydroxide CH:SOLVENT_B acetonitrile with 0.05 % ammonium hydroxide CH:SAMPLE_INJECTION 2μL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Samples were analyzed in positive and negative ion modes for both RP and HILIC MS:MS_COMMENTS chromatography. Spectral features were extracted from the raw data using MS:MS_COMMENTS Compound Discoverer v2.1 software. MS:CAPILLARY_TEMPERATURE 275 °C MS:SPRAY_VOLTAGE -2.8 kV MS:MS_RESULTS_FILE ST001172_AN001940_Results.txt UNITS:chromatographic peak area Has m/z:Neutral masses Has RT:No RT units:No RT data #END