#METABOLOMICS WORKBENCH wjZhang_echo_20221006_195331 DATATRACK_ID:3493 STUDY_ID:ST002341 ANALYSIS_ID:AN003824 PROJECT_ID:PR001503 VERSION 1 CREATED_ON November 11, 2022, 11:25 pm #PROJECT PR:PROJECT_TITLE Metabolomic analysis of colorectal cancer cells using mass spectrometry PR:PROJECT_SUMMARY Two pairs of human colorectal cancer cell lines with different metastatic PR:PROJECT_SUMMARY abilities (SW480 vs SW620, HT-29 vs COLO-205) were used to screen differential PR:PROJECT_SUMMARY metabolites. PR:INSTITUTE Nanjing Medical University PR:LAST_NAME Zhang PR:FIRST_NAME Wenjun PR:ADDRESS 101Longmian Avenue, Jiangning District, Nanjing 211166, P.R. China PR:EMAIL zwj941027369@163.com PR:PHONE +86-025-86868326 #STUDY ST:STUDY_TITLE Metabolomic analysis of colorectal cancer cells using mass spectrometry ST:STUDY_SUMMARY Colorectal cancer (CRC) is one of the most prevalent tumors, with a high ST:STUDY_SUMMARY mortality rate. Nearly half of CRC patients develop metastasis, which accounts ST:STUDY_SUMMARY for as many as 90% of CRC-related deaths. In the metastasis process, cancer ST:STUDY_SUMMARY cells exhibit altered dependency on specific metabolic pathways and some of the ST:STUDY_SUMMARY metabolites discovered might be useful as potential diagnostic biomarkers. To ST:STUDY_SUMMARY identify metabolic pathway dependencies in CRC metastasis, mass ST:STUDY_SUMMARY spectrometry-based untargeted metabolomic analysis was performed in two pairs of ST:STUDY_SUMMARY CRC cell lines with different metastatic abilities. Each pair of cell lines was ST:STUDY_SUMMARY comprised of primary and metastatic colorectal cancer cell lines (SW480 vs. ST:STUDY_SUMMARY SW620; HT-29 vs. COLO 205). Relative levels of intracellular metabolites ST:STUDY_SUMMARY distinguished high-metastatic CRC cells from low-metastatic CRC cells. ST:INSTITUTE Nanjing Medical University ST:LAST_NAME Zhang ST:FIRST_NAME Wenjun ST:ADDRESS 101Longmian Avenue, Jiangning District, Nanjing 211166, P.R. China ST:EMAIL zwj941027369@163.com ST:PHONE +86-025-86868326 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 BK_N_01 Blank_neg_01 Class:Blank RAW_FILE_NAME=Blank_neg_01.mzML SUBJECT_SAMPLE_FACTORS BK_N_02 Blank_neg_02 Class:Blank RAW_FILE_NAME=Blank_neg_02.mzML SUBJECT_SAMPLE_FACTORS BK_N_03 Blank_neg_03 Class:Blank RAW_FILE_NAME=Blank_neg_03.mzML SUBJECT_SAMPLE_FACTORS BK_N_04 Blank_neg_04 Class:Blank RAW_FILE_NAME=Blank_neg_04.mzML SUBJECT_SAMPLE_FACTORS BK_N_05 Blank_neg_05 Class:Blank RAW_FILE_NAME=Blank_neg_05.mzML SUBJECT_SAMPLE_FACTORS BK_N_06 Blank_neg_06 Class:Blank RAW_FILE_NAME=Blank_neg_06.mzML SUBJECT_SAMPLE_FACTORS BK_N_07 Blank_neg_07 Class:Blank RAW_FILE_NAME=Blank_neg_07.mzML SUBJECT_SAMPLE_FACTORS BK_N_08 Blank_neg_08 Class:Blank RAW_FILE_NAME=Blank_neg_08.mzML SUBJECT_SAMPLE_FACTORS QC_N_01 QC_neg_01 Class:QC RAW_FILE_NAME=QC_neg_01.mzML SUBJECT_SAMPLE_FACTORS QC_N_02 QC_neg_02 Class:QC RAW_FILE_NAME=QC_neg_02.mzML SUBJECT_SAMPLE_FACTORS QC_N_03 QC_neg_03 Class:QC RAW_FILE_NAME=QC_neg_03.mzML SUBJECT_SAMPLE_FACTORS QC_N_04 QC_neg_04 Class:QC RAW_FILE_NAME=QC_neg_04.mzML SUBJECT_SAMPLE_FACTORS QC_N_05 QC_neg_05 Class:QC RAW_FILE_NAME=QC_neg_05.mzML SUBJECT_SAMPLE_FACTORS QC_N_06 QC_neg_06 Class:QC RAW_FILE_NAME=QC_neg_06.mzML SUBJECT_SAMPLE_FACTORS QC_N_07 QC_neg_07 Class:QC RAW_FILE_NAME=QC_neg_07.mzML SUBJECT_SAMPLE_FACTORS QC_N_08 QC_neg_08 Class:QC RAW_FILE_NAME=QC_neg_08.mzML SUBJECT_SAMPLE_FACTORS S4_N_01 SW480_neg_01 Class:Low metastatic ability RAW_FILE_NAME=SW480_neg_01.mzML SUBJECT_SAMPLE_FACTORS S4_N_02 SW480_neg_02 Class:Low metastatic ability RAW_FILE_NAME=SW480_neg_02.mzML SUBJECT_SAMPLE_FACTORS S4_N_03 SW480_neg_03 Class:Low metastatic ability RAW_FILE_NAME=SW480_neg_03.mzML SUBJECT_SAMPLE_FACTORS S4_N_04 SW480_neg_04 Class:Low metastatic ability RAW_FILE_NAME=SW480_neg_04.mzML SUBJECT_SAMPLE_FACTORS S4_N_05 SW480_neg_05 Class:Low metastatic ability RAW_FILE_NAME=SW480_neg_05.mzML SUBJECT_SAMPLE_FACTORS S4_N_06 SW480_neg_06 Class:Low metastatic ability RAW_FILE_NAME=SW480_neg_06.mzML SUBJECT_SAMPLE_FACTORS S4_N_07 SW480_neg_07 Class:Low metastatic ability RAW_FILE_NAME=SW480_neg_07.mzML SUBJECT_SAMPLE_FACTORS S4_N_08 SW480_neg_08 Class:Low metastatic ability RAW_FILE_NAME=SW480_neg_08.mzML SUBJECT_SAMPLE_FACTORS S4_N_09 SW480_neg_09 Class:Low metastatic ability RAW_FILE_NAME=SW480_neg_09.mzML SUBJECT_SAMPLE_FACTORS S6_N_01 SW620_neg_01 Class:High metastatic ability RAW_FILE_NAME=SW620_neg_01.mzML SUBJECT_SAMPLE_FACTORS S6_N_02 SW620_neg_02 Class:High metastatic ability RAW_FILE_NAME=SW620_neg_02.mzML SUBJECT_SAMPLE_FACTORS S6_N_03 SW620_neg_03 Class:High metastatic ability RAW_FILE_NAME=SW620_neg_03.mzML SUBJECT_SAMPLE_FACTORS S6_N_04 SW620_neg_04 Class:High metastatic ability RAW_FILE_NAME=SW620_neg_04.mzML SUBJECT_SAMPLE_FACTORS S6_N_05 SW620_neg_05 Class:High metastatic ability RAW_FILE_NAME=SW620_neg_05.mzML SUBJECT_SAMPLE_FACTORS S6_N_06 SW620_neg_06 Class:High metastatic ability RAW_FILE_NAME=SW620_neg_06.mzML SUBJECT_SAMPLE_FACTORS S6_N_07 SW620_neg_07 Class:High metastatic ability RAW_FILE_NAME=SW620_neg_07.mzML SUBJECT_SAMPLE_FACTORS S6_N_08 SW620_neg_08 Class:High metastatic ability RAW_FILE_NAME=SW620_neg_08.mzML SUBJECT_SAMPLE_FACTORS S6_N_09 SW620_neg_09 Class:High metastatic ability RAW_FILE_NAME=SW620_neg_09.mzML SUBJECT_SAMPLE_FACTORS H_N_01 HT29_neg_01 Class:Low metastatic ability RAW_FILE_NAME=HT29_neg_01.mzML SUBJECT_SAMPLE_FACTORS H_N_02 HT29_neg_02 Class:Low metastatic ability RAW_FILE_NAME=HT29_neg_02.mzML SUBJECT_SAMPLE_FACTORS H_N_03 HT29_neg_03 Class:Low metastatic ability RAW_FILE_NAME=HT29_neg_03.mzML SUBJECT_SAMPLE_FACTORS H_N_04 HT29_neg_04 Class:Low metastatic ability RAW_FILE_NAME=HT29_neg_04.mzML SUBJECT_SAMPLE_FACTORS H_N_05 HT29_neg_05 Class:Low metastatic ability RAW_FILE_NAME=HT29_neg_05.mzML SUBJECT_SAMPLE_FACTORS H_N_06 HT29_neg_06 Class:Low metastatic ability RAW_FILE_NAME=HT29_neg_06.mzML SUBJECT_SAMPLE_FACTORS H_N_07 HT29_neg_07 Class:Low metastatic ability RAW_FILE_NAME=HT29_neg_07.mzML SUBJECT_SAMPLE_FACTORS H_N_08 HT29_neg_08 Class:Low metastatic ability RAW_FILE_NAME=HT29_neg_08.mzML SUBJECT_SAMPLE_FACTORS H_N_09 HT29_neg_09 Class:Low metastatic ability RAW_FILE_NAME=HT29_neg_09.mzML SUBJECT_SAMPLE_FACTORS C_N_01 COLO205_neg_01 Class:High metastatic ability RAW_FILE_NAME=COLO205_neg_01.mzML SUBJECT_SAMPLE_FACTORS C_N_02 COLO205_neg_02 Class:High metastatic ability RAW_FILE_NAME=COLO205_neg_02.mzML SUBJECT_SAMPLE_FACTORS C_N_03 COLO205_neg_03 Class:High metastatic ability RAW_FILE_NAME=COLO205_neg_03.mzML SUBJECT_SAMPLE_FACTORS C_N_04 COLO205_neg_04 Class:High metastatic ability RAW_FILE_NAME=COLO205_neg_04.mzML SUBJECT_SAMPLE_FACTORS C_N_05 COLO205_neg_05 Class:High metastatic ability RAW_FILE_NAME=COLO205_neg_05.mzML SUBJECT_SAMPLE_FACTORS C_N_06 COLO205_neg_06 Class:High metastatic ability RAW_FILE_NAME=COLO205_neg_06.mzML SUBJECT_SAMPLE_FACTORS C_N_07 COLO205_neg_07 Class:High metastatic ability RAW_FILE_NAME=COLO205_neg_07.mzML SUBJECT_SAMPLE_FACTORS C_N_08 COLO205_neg_08 Class:High metastatic ability RAW_FILE_NAME=COLO205_neg_08.mzML SUBJECT_SAMPLE_FACTORS C_N_09 COLO205_neg_09 Class:High metastatic ability RAW_FILE_NAME=COLO205_neg_09.mzML SUBJECT_SAMPLE_FACTORS BK_P_01 Blank_pos_01 Class:Blank RAW_FILE_NAME=Blank_pos_01.mzML SUBJECT_SAMPLE_FACTORS BK_P_02 Blank_pos_02 Class:Blank RAW_FILE_NAME=Blank_pos_02.mzML SUBJECT_SAMPLE_FACTORS BK_P_03 Blank_pos_03 Class:Blank RAW_FILE_NAME=Blank_pos_03.mzML SUBJECT_SAMPLE_FACTORS BK_P_04 Blank_pos_04 Class:Blank RAW_FILE_NAME=Blank_pos_04.mzML SUBJECT_SAMPLE_FACTORS BK_P_05 Blank_pos_05 Class:Blank RAW_FILE_NAME=Blank_pos_05.mzML SUBJECT_SAMPLE_FACTORS BK_P_06 Blank_pos_06 Class:Blank RAW_FILE_NAME=Blank_pos_06.mzML SUBJECT_SAMPLE_FACTORS BK_P_07 Blank_pos_07 Class:Blank RAW_FILE_NAME=Blank_pos_07.mzML SUBJECT_SAMPLE_FACTORS BK_P_08 Blank_pos_08 Class:Blank RAW_FILE_NAME=Blank_pos_08.mzML SUBJECT_SAMPLE_FACTORS QC_P_01 QC_pos_01 Class:QC RAW_FILE_NAME=QC_pos_01.mzML SUBJECT_SAMPLE_FACTORS QC_P_02 QC_pos_02 Class:QC RAW_FILE_NAME=QC_pos_02.mzML SUBJECT_SAMPLE_FACTORS QC_P_03 QC_pos_03 Class:QC RAW_FILE_NAME=QC_pos_03.mzML SUBJECT_SAMPLE_FACTORS QC_P_04 QC_pos_04 Class:QC RAW_FILE_NAME=QC_pos_04.mzML SUBJECT_SAMPLE_FACTORS QC_P_05 QC_pos_05 Class:QC RAW_FILE_NAME=QC_pos_05.mzML SUBJECT_SAMPLE_FACTORS QC_P_06 QC_pos_06 Class:QC RAW_FILE_NAME=QC_pos_06.mzML SUBJECT_SAMPLE_FACTORS QC_P_07 QC_pos_07 Class:QC RAW_FILE_NAME=QC_pos_07.mzML SUBJECT_SAMPLE_FACTORS QC_P_08 QC_pos_08 Class:QC RAW_FILE_NAME=QC_pos_08.mzML SUBJECT_SAMPLE_FACTORS S4_P_01 SW480_pos_01 Class:Low metastatic ability RAW_FILE_NAME=SW480_pos_01.mzML SUBJECT_SAMPLE_FACTORS S4_P_02 SW480_pos_02 Class:Low metastatic ability RAW_FILE_NAME=SW480_pos_02.mzML SUBJECT_SAMPLE_FACTORS S4_P_03 SW480_pos_03 Class:Low metastatic ability RAW_FILE_NAME=SW480_pos_03.mzML SUBJECT_SAMPLE_FACTORS S4_P_04 SW480_pos_04 Class:Low metastatic ability RAW_FILE_NAME=SW480_pos_04.mzML SUBJECT_SAMPLE_FACTORS S4_P_05 SW480_pos_05 Class:Low metastatic ability RAW_FILE_NAME=SW480_pos_05.mzML SUBJECT_SAMPLE_FACTORS S4_P_06 SW480_pos_06 Class:Low metastatic ability RAW_FILE_NAME=SW480_pos_06.mzML SUBJECT_SAMPLE_FACTORS S4_P_07 SW480_pos_07 Class:Low metastatic ability RAW_FILE_NAME=SW480_pos_07.mzML SUBJECT_SAMPLE_FACTORS S4_P_08 SW480_pos_08 Class:Low metastatic ability RAW_FILE_NAME=SW480_pos_08.mzML SUBJECT_SAMPLE_FACTORS S4_P_09 SW480_pos_09 Class:Low metastatic ability RAW_FILE_NAME=SW480_pos_09.mzML SUBJECT_SAMPLE_FACTORS S6_P_01 SW620_pos_01 Class:High metastatic ability RAW_FILE_NAME=SW620_pos_01.mzML SUBJECT_SAMPLE_FACTORS S6_P_02 SW620_pos_02 Class:High metastatic ability RAW_FILE_NAME=SW620_pos_02.mzML SUBJECT_SAMPLE_FACTORS S6_P_03 SW620_pos_03 Class:High metastatic ability RAW_FILE_NAME=SW620_pos_03.mzML SUBJECT_SAMPLE_FACTORS S6_P_04 SW620_pos_04 Class:High metastatic ability RAW_FILE_NAME=SW620_pos_04.mzML SUBJECT_SAMPLE_FACTORS S6_P_05 SW620_pos_05 Class:High metastatic ability RAW_FILE_NAME=SW620_pos_05.mzML SUBJECT_SAMPLE_FACTORS S6_P_06 SW620_pos_06 Class:High metastatic ability RAW_FILE_NAME=SW620_pos_06.mzML SUBJECT_SAMPLE_FACTORS S6_P_07 SW620_pos_07 Class:High metastatic ability RAW_FILE_NAME=SW620_pos_07.mzML SUBJECT_SAMPLE_FACTORS S6_P_08 SW620_pos_08 Class:High metastatic ability RAW_FILE_NAME=SW620_pos_08.mzML SUBJECT_SAMPLE_FACTORS S6_P_09 SW620_pos_09 Class:High metastatic ability RAW_FILE_NAME=SW620_pos_09.mzML SUBJECT_SAMPLE_FACTORS H_P_01 HT29_pos_01 Class:Low metastatic ability RAW_FILE_NAME=HT29_pos_01.mzML SUBJECT_SAMPLE_FACTORS H_P_02 HT29_pos_02 Class:Low metastatic ability RAW_FILE_NAME=HT29_pos_02.mzML SUBJECT_SAMPLE_FACTORS H_P_03 HT29_pos_03 Class:Low metastatic ability RAW_FILE_NAME=HT29_pos_03.mzML SUBJECT_SAMPLE_FACTORS H_P_04 HT29_pos_04 Class:Low metastatic ability RAW_FILE_NAME=HT29_pos_04.mzML SUBJECT_SAMPLE_FACTORS H_P_05 HT29_pos_05 Class:Low metastatic ability RAW_FILE_NAME=HT29_pos_05.mzML SUBJECT_SAMPLE_FACTORS H_P_06 HT29_pos_06 Class:Low metastatic ability RAW_FILE_NAME=HT29_pos_06.mzML SUBJECT_SAMPLE_FACTORS H_P_07 HT29_pos_07 Class:Low metastatic ability RAW_FILE_NAME=HT29_pos_07.mzML SUBJECT_SAMPLE_FACTORS H_P_08 HT29_pos_08 Class:Low metastatic ability RAW_FILE_NAME=HT29_pos_08.mzML SUBJECT_SAMPLE_FACTORS H_P_09 HT29_pos_09 Class:Low metastatic ability RAW_FILE_NAME=HT29_pos_09.mzML SUBJECT_SAMPLE_FACTORS C_P_01 COLO205_pos_01 Class:High metastatic ability RAW_FILE_NAME=COLO205_pos_01.mzML SUBJECT_SAMPLE_FACTORS C_P_02 COLO205_pos_02 Class:High metastatic ability RAW_FILE_NAME=COLO205_pos_02.mzML SUBJECT_SAMPLE_FACTORS C_P_03 COLO205_pos_03 Class:High metastatic ability RAW_FILE_NAME=COLO205_pos_03.mzML SUBJECT_SAMPLE_FACTORS C_P_04 COLO205_pos_04 Class:High metastatic ability RAW_FILE_NAME=COLO205_pos_04.mzML SUBJECT_SAMPLE_FACTORS C_P_05 COLO205_pos_05 Class:High metastatic ability RAW_FILE_NAME=COLO205_pos_05.mzML SUBJECT_SAMPLE_FACTORS C_P_06 COLO205_pos_06 Class:High metastatic ability RAW_FILE_NAME=COLO205_pos_06.mzML SUBJECT_SAMPLE_FACTORS C_P_07 COLO205_pos_07 Class:High metastatic ability RAW_FILE_NAME=COLO205_pos_07.mzML SUBJECT_SAMPLE_FACTORS C_P_08 COLO205_pos_08 Class:High metastatic ability RAW_FILE_NAME=COLO205_pos_08.mzML SUBJECT_SAMPLE_FACTORS C_P_09 COLO205_pos_09 Class:High metastatic ability RAW_FILE_NAME=COLO205_pos_09.mzML #COLLECTION CO:COLLECTION_SUMMARY Two hours before metabolite extraction, the cell culture medium was replaced CO:COLLECTION_SUMMARY with fresh medium. After discarding the medium in each culture dish, the cells CO:COLLECTION_SUMMARY were quickly rinsed twice with cold isotonic saline (0.9% NaCl [w/v], 4 °C). CO:COLLECTION_SUMMARY Water (1.5 mL) was added to each dish, and then the dishes were stored in a CO:COLLECTION_SUMMARY freezer (−80 °C) for 20 min before extraction. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY SW480 cells and SW620 cells were cultured in DMEM medium supplemented with 10% TR:TREATMENT_SUMMARY FBS at 37 °C under a 5% CO2 atmosphere, HT-29 and COLO 205 cells were cultured TR:TREATMENT_SUMMARY in RPMI 1640 medium supplemented with 10% FBS at 37 °C under a 5% CO2 TR:TREATMENT_SUMMARY atmosphere. TR:CELL_MEDIA DMEM medium supplemented with 10% FBS is for SW480 cells and SW620 cells, while TR:CELL_MEDIA RPMI 1640 medium supplemented with 10% FBS is for HT-29 and COLO 205 cells. TR:CELL_ENVIR_COND at 37 °C under a 5% CO2 atmosphere TR:CELL_HARVESTING After discarding the medium in each culture dish, the cells were quickly rinsed TR:CELL_HARVESTING twice with cold isotonic saline (0.9% NaCl [w/v], 4 °C). Water (1.5 mL) was TR:CELL_HARVESTING added to each dish, and then the dishes were stored in a freezer (−80 °C) for TR:CELL_HARVESTING 20 min before extraction. Then, cells were collected by scraping with a cell TR:CELL_HARVESTING scraper. TR:CELL_PCT_CONFLUENCE 70%-80% #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cell lysates (20 μL) were removed for a protein assay, and the remaining cell SP:SAMPLEPREP_SUMMARY lysates were extracted by the addition of 4.5 mL of methanol containing 3 μg of SP:SAMPLEPREP_SUMMARY internal standard (acetaminophen). The cells were completely collected with a SP:SAMPLEPREP_SUMMARY cell scraper. Finally, the suspension of cell debris in each dish was SP:SAMPLEPREP_SUMMARY transferred to an Eppendorf tube, vigorously vortexed for 5 min, and then SP:SAMPLEPREP_SUMMARY centrifuged at 10,000×g for 10 min at 4 °C. The supernatant was transferred to SP:SAMPLEPREP_SUMMARY another tube and evaporated to dryness in a Refrigerated CentriVap Benchtop SP:SAMPLEPREP_SUMMARY Vacuum Concentrator (Labconco Corporation, Kansas City, MO). The pellets were SP:SAMPLEPREP_SUMMARY reconstituted with methanol-water (75:25) and centrifuged twice at 12,000×g for SP:SAMPLEPREP_SUMMARY 15 min at 4 °C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME UPLC Ultimate 3000 system CH:COLUMN_NAME Phenomenex Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm) CH:FLOW_GRADIENT 10% B (0 min) → 30% B (1 min) → 95% B (19 min) → 95% B (20 min) CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40 °C CH:SOLVENT_A Water containing 0.1% formic acid (v/v) CH:SOLVENT_B Acetonitrile containing 0.1% formic acid (v/v) CH:INJECTION_TEMPERATURE 4 °C #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 POSITIVE MS:MS_COMMENTS All operations and acquisitions were controlled by Xcalibur 2.0.7 (Thermo Fisher MS:MS_COMMENTS Scientific, Bremen, Germany).UPLC-HRMS data were analyzed using MS-DIAL software MS:MS_COMMENTS after conversion to mzML file format using MSConvert (Version 3.0, MS:MS_COMMENTS ProteiWizard). MS:CAPILLARY_TEMPERATURE 300 °C MS:CAPILLARY_VOLTAGE +3.5 kV MS:MS_RESULTS_FILE ST002341_AN003824_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END