#METABOLOMICS WORKBENCH eschwiebert_20241007_125033 DATATRACK_ID:5268 STUDY_ID:ST003564 ANALYSIS_ID:AN005856 PROJECT_ID:PR002198 VERSION 1 CREATED_ON 11-07-2024 #PROJECT PR:PROJECT_TITLE Metabolomics of Papanicolaou Tests for the Discovery of Ovarian Cancer PR:PROJECT_TITLE Biomarkers PR:PROJECT_TYPE Untargeted Metabolomics PR:PROJECT_SUMMARY Ovarian cancer (OC) remains one of the most lethal cancers among female PR:PROJECT_SUMMARY populations due to the vast majority of cases going undiagnosed until later PR:PROJECT_SUMMARY stages (stage III, IV). Early detection and treatment of this malignancy PR:PROJECT_SUMMARY provides the best prognosis, but the lack of accurate and sensitive screening PR:PROJECT_SUMMARY tools combined with the presence of ambiguous symptoms hinders these diagnoses. PR:PROJECT_SUMMARY In contrast, screening for cervical cancer via Papanicolaou (Pap) tests is a PR:PROJECT_SUMMARY widespread practice with use spanning the past several decades, reducing this PR:PROJECT_SUMMARY cancerâs morbidity and mortality rates. Two types of Pap smear tests exist: a PR:PROJECT_SUMMARY conventional method where cells from the ectocervix are stained on a glass slide PR:PROJECT_SUMMARY and a liquid-based cytology method. Interestingly, previous studies show PR:PROJECT_SUMMARY evidence of OC cells in Pap tests, suggesting that lipids shed from ovarian PR:PROJECT_SUMMARY tumors may end up in the cervix. The goal of this study is to evaluate the PR:PROJECT_SUMMARY practicality of using liquid-based Pap tests as biospecimens for OC PR:PROJECT_SUMMARY screening-related metabolic studies. Cell pellets from liquid-based Pap tests PR:PROJECT_SUMMARY from 29 healthy women were analyzed via ultra-high performance liquid PR:PROJECT_SUMMARY chromatography mass spectrometry (UHPLC-MS). This approach facilitated the PR:PROJECT_SUMMARY detection and annotation of 453 unique lipids across 20 lipid subclasses, PR:PROJECT_SUMMARY including ceramides, triacylglycerols, and hexosylceramides. These results PR:PROJECT_SUMMARY demonstrated the feasibility of using an MS-based approach to analyze residual PR:PROJECT_SUMMARY Pap test samples for the discovery of OC-related lipid biomarkers with the goal PR:PROJECT_SUMMARY of detecting OC at early stages of the disease. PR:INSTITUTE Georgia Institute of Technology PR:DEPARTMENT Chemistry and Biochemistry PR:LABORATORY Fernandez Lab PR:LAST_NAME Schwiebert PR:FIRST_NAME Elisabeth PR:ADDRESS 311 Ferst Dr NW, Atlanta, Georgia, 30318, USA PR:EMAIL eschwiebert3@gatech.edu PR:PHONE 4043854432 PR:FUNDING_SOURCE National Institutes of Health's National Center for Advancing Translational PR:FUNDING_SOURCE Sciences PR:DOI http://dx.doi.org/10.21228/M8HC23 #STUDY ST:STUDY_TITLE Metabolomics of Papanicolaou Tests for the Discovery of Ovarian Cancer ST:STUDY_TITLE Biomarkers ST:STUDY_TYPE Metabolomics Studies on Papanicolaou Tests ST:STUDY_SUMMARY Ovarian cancer (OC) remains one of the most lethal cancers among female ST:STUDY_SUMMARY populations due to the vast majority of cases going undiagnosed until later ST:STUDY_SUMMARY stages (stage III, IV). Early detection and treatment of this malignancy ST:STUDY_SUMMARY provides the best prognosis, but the lack of accurate and sensitive screening ST:STUDY_SUMMARY tools combined with the presence of ambiguous symptoms hinders these diagnoses. ST:STUDY_SUMMARY In contrast, screening for cervical cancer via Papanicolaou (Pap) tests is a ST:STUDY_SUMMARY widespread practice with use spanning the past several decades, reducing this ST:STUDY_SUMMARY cancerâs morbidity and mortality rates. Two types of Pap smear tests exist: a ST:STUDY_SUMMARY conventional method where cells from the ectocervix are stained on a glass slide ST:STUDY_SUMMARY and a liquid-based cytology method. Interestingly, previous studies show ST:STUDY_SUMMARY evidence of OC cells in Pap tests, suggesting that lipids shed from ovarian ST:STUDY_SUMMARY tumors may end up in the cervix. The goal of this study is to evaluate the ST:STUDY_SUMMARY practicality of using liquid-based Pap tests as biospecimens for OC ST:STUDY_SUMMARY screening-related metabolic studies. Cell pellets, extracted from residual Pap ST:STUDY_SUMMARY test fluid (RPF), from liquid-based Pap tests from 29 healthy women were ST:STUDY_SUMMARY analyzed via ultra-high performance liquid chromatography mass spectrometry ST:STUDY_SUMMARY (UHPLC-MS). This approach facilitated the detection and annotation of 453 unique ST:STUDY_SUMMARY lipids across 20 lipid subclasses, including ceramides, triacylglycerols, and ST:STUDY_SUMMARY hexosylceramides. These results demonstrated the feasibility of using an ST:STUDY_SUMMARY MS-based approach to analyze residual Pap test samples for the discovery of ST:STUDY_SUMMARY OC-related lipid biomarkers with the goal of detecting OC at early stages of the ST:STUDY_SUMMARY disease. ST:INSTITUTE Georgia Institute of Technology ST:DEPARTMENT Chemistry and Biochemistry ST:LABORATORY Fernandez Lab ST:LAST_NAME Schwiebert ST:FIRST_NAME Elisabeth ST:ADDRESS 311 Ferst Dr NW, Atlanta, Georgia, 30318, USA ST:EMAIL eschwiebert3@gatech.edu ST:PHONE 4043854432 ST:SUBMIT_DATE 2024-10-07 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 50+ SU:GENDER Female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Blank-01 blank1 Sample source:Blank | Sample Type:Blank RAW_FILE_NAME=blank1.mzML SUBJECT_SAMPLE_FACTORS Blank-02 blank2 Sample source:Blank | Sample Type:Blank RAW_FILE_NAME=blank2.mzML SUBJECT_SAMPLE_FACTORS Blank-03 blank3 Sample source:Blank | Sample Type:Blank RAW_FILE_NAME=blank3.mzML SUBJECT_SAMPLE_FACTORS Blank-04 blank4 Sample source:Blank | Sample Type:Blank RAW_FILE_NAME=blank4.mzML SUBJECT_SAMPLE_FACTORS Pellet-10 10 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=10.mzML SUBJECT_SAMPLE_FACTORS Pellet-101 101 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=101.mzML SUBJECT_SAMPLE_FACTORS Pellet-105 105 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=105.mzML SUBJECT_SAMPLE_FACTORS Pellet-111 111 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=111.mzML SUBJECT_SAMPLE_FACTORS Pellet-113 113 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=113.mzML SUBJECT_SAMPLE_FACTORS Pellet-119 119 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=119.mzML SUBJECT_SAMPLE_FACTORS Pellet-132 132 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=132.mzML SUBJECT_SAMPLE_FACTORS Pellet-14 14 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=14.mzML SUBJECT_SAMPLE_FACTORS Pellet-151 151 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=151.mzML SUBJECT_SAMPLE_FACTORS Pellet-154 154 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=154.mzML SUBJECT_SAMPLE_FACTORS Pellet-160 160 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=160.mzML SUBJECT_SAMPLE_FACTORS Pellet-18 18 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=18.mzML SUBJECT_SAMPLE_FACTORS Pellet-20 20 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=20.mzML SUBJECT_SAMPLE_FACTORS Pellet-26 26 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=26.mzML SUBJECT_SAMPLE_FACTORS Pellet-34 34 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=34.mzML SUBJECT_SAMPLE_FACTORS Pellet-35 35 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=35.mzML SUBJECT_SAMPLE_FACTORS Pellet-43 43 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=43.mzML SUBJECT_SAMPLE_FACTORS Pellet-49 49 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=49.mzML SUBJECT_SAMPLE_FACTORS Pellet-06 6 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=6.mzML SUBJECT_SAMPLE_FACTORS Pellet-61 61 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=61.mzML SUBJECT_SAMPLE_FACTORS Pellet-69 69 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=69.mzML SUBJECT_SAMPLE_FACTORS Pellet-73 73 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=73.mzML SUBJECT_SAMPLE_FACTORS Pellet-74 74 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=74.mzML SUBJECT_SAMPLE_FACTORS Pellet-76 76 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=76.mzML SUBJECT_SAMPLE_FACTORS Pellet-77 77 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=77.mzML SUBJECT_SAMPLE_FACTORS Pellet-84 84 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=84.mzML SUBJECT_SAMPLE_FACTORS Pellet-93 93 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=93.mzML SUBJECT_SAMPLE_FACTORS Pellet-96 96 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=96.mzML SUBJECT_SAMPLE_FACTORS Pellet-99 99 Sample source:Cell Pellet (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=99.mzML SUBJECT_SAMPLE_FACTORS QC0 QC0 Sample source:QC | Sample Type:Pool RAW_FILE_NAME=QC0.mzML SUBJECT_SAMPLE_FACTORS QC00 QC00 Sample source:QC | Sample Type:Pool RAW_FILE_NAME=QC00.mzML SUBJECT_SAMPLE_FACTORS QC1 QC1 Sample source:QC | Sample Type:Pool RAW_FILE_NAME=QC1.mzML SUBJECT_SAMPLE_FACTORS QC2 QC2 Sample source:QC | Sample Type:Pool RAW_FILE_NAME=QC2.mzML SUBJECT_SAMPLE_FACTORS QC3 QC3 Sample source:QC | Sample Type:Pool RAW_FILE_NAME=QC3.mzML SUBJECT_SAMPLE_FACTORS QC4 QC4 Sample source:QC | Sample Type:Pool RAW_FILE_NAME=QC4.mzML SUBJECT_SAMPLE_FACTORS QC5 QC5 Sample source:QC | Sample Type:Pool RAW_FILE_NAME=QC5.mzML SUBJECT_SAMPLE_FACTORS Supernatent-27-01 27-01 Sample source:Supernatant (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=Supernatent-27-01.mzML SUBJECT_SAMPLE_FACTORS Supernatent-27-02 27-02 Sample source:Supernatant (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=Supernatent-27-02.mzML SUBJECT_SAMPLE_FACTORS Supernatent-27-03 27-03 Sample source:Supernatant (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=Supernatent-27-03.mzML SUBJECT_SAMPLE_FACTORS Supernatent-45-01 45-01 Sample source:Supernatant (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=Supernatent-45-01.mzML SUBJECT_SAMPLE_FACTORS Supernatent-45-02 45-02 Sample source:Supernatant (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=Supernatent-45-02.mzML SUBJECT_SAMPLE_FACTORS Supernatent-45-03 45-03 Sample source:Supernatant (extracted from RPF) | Sample Type:Control RAW_FILE_NAME=Supernatent-45-03.mzML #COLLECTION CO:COLLECTION_SUMMARY Samples were vortexed and centrifuged to separate samples' cell pellet and CO:COLLECTION_SUMMARY supernatant layers before undergoing separate extraction methods. The residual CO:COLLECTION_SUMMARY BD SurePathTM liquid-based Pap test samples (~2 mL each) were transferred to 2 CO:COLLECTION_SUMMARY mL microcentrifuge tubes. Samples were vortexed for 10s followed by CO:COLLECTION_SUMMARY centrifugation at approximately 7,130 x g (5000 rpm) for 5 minutes to pellet the CO:COLLECTION_SUMMARY cells. Supernatants were removed and saved for further analysis. CO:SAMPLE_TYPE Supernatants and cell pellets extracted from residual Pap test fluid #TREATMENT TR:TREATMENT_SUMMARY Residual Papanicolaou tests were obtained from the University of Minnesota TR:TREATMENT_SUMMARY BioNext Tissue Procurement Facility and stored at 4-5C prior to analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cell pellets, extracted from residual Pap test fluid, were dried in a SpeedVac SP:SAMPLEPREP_SUMMARY and weighed. Six hundred µL of IPA and 0.2 grams of 500 µm glass beads were SP:SAMPLEPREP_SUMMARY then added to each cell pellet, followed by homogenization in a Tissuelyser. SP:SAMPLEPREP_SUMMARY Samples were then re-dried in a SpeedVac, and the metabolites were extracted SP:SAMPLEPREP_SUMMARY with a biphasic solution comprised of 600 µL chloroform, 600 µL methanol, and SP:SAMPLEPREP_SUMMARY 300 µL water. Extracts were sonicated for 5 minutes and then centrifuged at SP:SAMPLEPREP_SUMMARY 21,100 x g (14,800 rpm) for 7 minutes. The chloroform extract from each sample SP:SAMPLEPREP_SUMMARY was transferred to a 1.5 mL microcentrifuge tube and dried in the SpeedVac. An SP:SAMPLEPREP_SUMMARY extraction mixture was prepared by mixing 50 µL of the isotopically labeled SP:SAMPLEPREP_SUMMARY internal standards mixture with 3000 µL of IPA. Eighty microliters of this SP:SAMPLEPREP_SUMMARY mixture were added to each of the dried chloroform extracts. After IPA SP:SAMPLEPREP_SUMMARY extraction, samples underwent a second cycle of sonication for 5 minutes and SP:SAMPLEPREP_SUMMARY centrifugation at 21,100 x g (14,800 rpm) for 7 minutes. Cell pellet extracts SP:SAMPLEPREP_SUMMARY and supernatant extracts were then transferred to LC-vials for UHPLC-MS SP:SAMPLEPREP_SUMMARY analysis. A blank sample was prepared with LC-MS grade IPA and underwent the SP:SAMPLEPREP_SUMMARY same preparation process as the samples. A pooled quality control (QC) sample SP:SAMPLEPREP_SUMMARY was prepared by mixing 3 µL aliquots of each of the sample extracts. Samples SP:SAMPLEPREP_SUMMARY were stored at 4-5 ºC until analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Negative Ion Mode; Solvent A: 10 mM Ammonium Acetate in Water/Acetonitrile CH:CHROMATOGRAPHY_SUMMARY (40:60 v/v); Solvent B: 10 mM Ammonium Acetate in 2-Isopropanol/Acetonitrile CH:CHROMATOGRAPHY_SUMMARY (90:10 v/v) CH:METHODS_FILENAME Pap_Negative_MS1.pdf CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Accucore C30 (150 x 2.1mm,2.6um) CH:COLUMN_TEMPERATURE 50 CH:FLOW_GRADIENT 0 min: 20% B, 0-1 min: 20% B, 1-5 min: 60% B, 5-5.5 min: 70% B, 5.5-8 min: 85% CH:FLOW_GRADIENT B, 8-8.2: 90% B, 8.2-10.5: 100% B, 10.5-10.7: 100% B, 10.7-12: 20% B CH:FLOW_RATE 0.400 mL/min CH:SOLVENT_A 40% Water/60% Acetonitrile; 10 mM Ammonium Acetate in CH:SOLVENT_B 90% 2-Isopropanol/10% Acetonitrile; 10 mM Ammonium Acetate CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS AN:ACQUISITION_PARAMETERS_FILE Pap_Negative_MS1.pdf; Pap_Negative_MS2.pdf #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X Tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS MS1 Analysis. Raw data files were processed using Compound Discoverer v3.3. MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST003564_AN005856_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END