#METABOLOMICS WORKBENCH Linyan_20231218_233331 DATATRACK_ID:4537 STUDY_ID:ST003025 ANALYSIS_ID:AN004960 PROJECT_ID:PR001876 VERSION 1 CREATED_ON December 22, 2023, 12:01 am #PROJECT PR:PROJECT_TITLE NMR- and MS-based omics reveal characteristic metabolome atlas and optimize PR:PROJECT_TITLE biofluid earlydiagnostic biomarkers for esophageal squamous cell carcinoma PR:PROJECT_SUMMARY Metabolic changes precede malignant histology. However, it remains unclear PR:PROJECT_SUMMARY whether detectable characteristic metabolome exists in esophageal squamous cell PR:PROJECT_SUMMARY carcinoma (ESCC) tissues and biofluids for early diagnosis. We conducted NMR- PR:PROJECT_SUMMARY and MS-based metabolomics on 1,153 matched ESCC tissues, normal mucosae, pre- PR:PROJECT_SUMMARY and one-week post-operative sera and urines from 560 participants across three PR:PROJECT_SUMMARY hospitals, with machine learning, logistic regression and WGCNA. Aberrations in PR:PROJECT_SUMMARY 'alanine, aspartate and glutamate metabolism' proved to be prevalent throughout PR:PROJECT_SUMMARY the ESCC evolution, and were reflected in 16 serum and 10 urine metabolic PR:PROJECT_SUMMARY signatures that were consistently identified by NMR and MS in both discovery and PR:PROJECT_SUMMARY validation sets. NMR-based simplified panels of any five serum or urine PR:PROJECT_SUMMARY metabolites outperformed clinical serological tumor markers (AUC = 0.984 and PR:PROJECT_SUMMARY 0.930, respectively), and were effective in distinguishing early-stage ESCC in PR:PROJECT_SUMMARY test set (serum accuracy = 0.994, urine accuracy = 0.879). Collectively, PR:PROJECT_SUMMARY NMR-based biofluid screening can reveal characteristic metabolic events of ESCC PR:PROJECT_SUMMARY and be feasible for early detection (ChiCTR2300073613). PR:INSTITUTE Radiology Department, Second Affiliated Hospital, Shantou University Medical PR:INSTITUTE College, Shantou PR:LAST_NAME Lin PR:FIRST_NAME Yan PR:ADDRESS No. 69, Dongxia North Road, Shantou, Guangdong, China PR:EMAIL 994809889@qq.com PR:PHONE +86 18823992148 #STUDY ST:STUDY_TITLE NMR- and MS-based omics reveal characteristic metabolome atlas and optimize ST:STUDY_TITLE biofluid earlydiagnostic biomarkers for esophageal squamous cell carcinoma ST:STUDY_TITLE (part-Ⅴ) ST:STUDY_SUMMARY Metabolic changes precede malignant histology. However, it remains unclear ST:STUDY_SUMMARY whether detectable characteristic metabolome exists in esophageal squamous cell ST:STUDY_SUMMARY carcinoma (ESCC) tissues and biofluids for early diagnosis. We conducted NMR- ST:STUDY_SUMMARY and MS-based metabolomics on 1,153 matched ESCC tissues, normal mucosae, pre- ST:STUDY_SUMMARY and one-week post-operative sera and urines from 560 participants across three ST:STUDY_SUMMARY hospitals, with machine learning, logistic regression and WGCNA. Aberrations in ST:STUDY_SUMMARY 'alanine, aspartate and glutamate metabolism' proved to be prevalent throughout ST:STUDY_SUMMARY the ESCC evolution, and were reflected in 16 serum and 10 urine metabolic ST:STUDY_SUMMARY signatures that were consistently identified by NMR and MS in both discovery and ST:STUDY_SUMMARY validation sets. NMR-based simplified panels of any five serum or urine ST:STUDY_SUMMARY metabolites outperformed clinical serological tumor markers (AUC = 0.984 and ST:STUDY_SUMMARY 0.930, respectively), and were effective in distinguishing early-stage ESCC in ST:STUDY_SUMMARY test set (serum accuracy = 0.994, urine accuracy = 0.879). Collectively, ST:STUDY_SUMMARY NMR-based biofluid screening can reveal characteristic metabolic events of ESCC ST:STUDY_SUMMARY and be feasible for early detection (ChiCTR2300073613). ST:INSTITUTE Radiology Department, Second Affiliated Hospital, Shantou University Medical ST:INSTITUTE College, Shantou ST:LAST_NAME Lin ST:FIRST_NAME Yan ST:ADDRESS No. 69, Dongxia North Road, Shantou, Guangdong, China ST:EMAIL 994809889@qq.com ST:PHONE +86 18823992148 #SUBJECT SU:SUBJECT_TYPE Human 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 - 1 Fator:Early stage ESCC RAW_FILE_NAME=1.CDF SUBJECT_SAMPLE_FACTORS - 2 Fator:Early stage ESCC RAW_FILE_NAME=2.CDF SUBJECT_SAMPLE_FACTORS - 3 Fator:Early stage ESCC RAW_FILE_NAME=3.CDF SUBJECT_SAMPLE_FACTORS - 4 Fator:Early stage ESCC RAW_FILE_NAME=4.CDF SUBJECT_SAMPLE_FACTORS - 5 Fator:Early stage ESCC RAW_FILE_NAME=5.CDF SUBJECT_SAMPLE_FACTORS - 6 Fator:Early stage ESCC RAW_FILE_NAME=6.CDF SUBJECT_SAMPLE_FACTORS - 7 Fator:Early stage ESCC RAW_FILE_NAME=7.CDF SUBJECT_SAMPLE_FACTORS - 8 Fator:Early stage ESCC RAW_FILE_NAME=8.CDF SUBJECT_SAMPLE_FACTORS - 9 Fator:Early stage ESCC RAW_FILE_NAME=9.CDF SUBJECT_SAMPLE_FACTORS - 10 Fator:Early stage ESCC RAW_FILE_NAME=10.CDF SUBJECT_SAMPLE_FACTORS - 11 Fator:Early stage ESCC RAW_FILE_NAME=11.CDF SUBJECT_SAMPLE_FACTORS - 12 Fator:Early stage ESCC RAW_FILE_NAME=12.CDF SUBJECT_SAMPLE_FACTORS - 13 Fator:Early stage ESCC RAW_FILE_NAME=13.CDF SUBJECT_SAMPLE_FACTORS - 14 Fator:Early stage ESCC RAW_FILE_NAME=14.CDF SUBJECT_SAMPLE_FACTORS - 15 Fator:Early stage ESCC RAW_FILE_NAME=15.CDF SUBJECT_SAMPLE_FACTORS - 16 Fator:Early stage ESCC RAW_FILE_NAME=16.CDF SUBJECT_SAMPLE_FACTORS - 17 Fator:Normal tissue RAW_FILE_NAME=17.CDF SUBJECT_SAMPLE_FACTORS - 18 Fator:Normal tissue RAW_FILE_NAME=18.CDF SUBJECT_SAMPLE_FACTORS - 19 Fator:Normal tissue RAW_FILE_NAME=19.CDF SUBJECT_SAMPLE_FACTORS - 20 Fator:Normal tissue RAW_FILE_NAME=20.CDF SUBJECT_SAMPLE_FACTORS - 21 Fator:Normal tissue RAW_FILE_NAME=21.CDF SUBJECT_SAMPLE_FACTORS - 22 Fator:Normal tissue RAW_FILE_NAME=22.CDF SUBJECT_SAMPLE_FACTORS - 23 Fator:Normal tissue RAW_FILE_NAME=23.CDF SUBJECT_SAMPLE_FACTORS - 24 Fator:Normal tissue RAW_FILE_NAME=24.CDF SUBJECT_SAMPLE_FACTORS - 25 Fator:Normal tissue RAW_FILE_NAME=25.CDF SUBJECT_SAMPLE_FACTORS - 26 Fator:Normal tissue RAW_FILE_NAME=26.CDF SUBJECT_SAMPLE_FACTORS - 27 Fator:Normal tissue RAW_FILE_NAME=27.CDF SUBJECT_SAMPLE_FACTORS - 28 Fator:Normal tissue RAW_FILE_NAME=28.CDF SUBJECT_SAMPLE_FACTORS - 29 Fator:Normal tissue RAW_FILE_NAME=29.CDF SUBJECT_SAMPLE_FACTORS - 30 Fator:Normal tissue RAW_FILE_NAME=30.CDF SUBJECT_SAMPLE_FACTORS - 31 Fator:Normal tissue RAW_FILE_NAME=31.CDF SUBJECT_SAMPLE_FACTORS - 32 Fator:Normal tissue RAW_FILE_NAME=32.CDF #COLLECTION CO:COLLECTION_SUMMARY Tissue samples, including tumor and normal areas 5 cm away, were obtained under CO:COLLECTION_SUMMARY the guidance of experienced pathologists without compromising the patients' CO:COLLECTION_SUMMARY pathology examinations. The collected tissue was rinsed with PBS to avoid CO:COLLECTION_SUMMARY contamination, excess moisture was removed, and it was rapidly frozen in liquid CO:COLLECTION_SUMMARY nitrogen to arrest enzymatic or chemical reactions. Samples were stored at CO:COLLECTION_SUMMARY −80°C until metabolite extraction. CO:SAMPLE_TYPE Tissue #TREATMENT TR:TREATMENT_SUMMARY None #SAMPLEPREP SP:SAMPLEPREP_SUMMARY GC-MS Analysis mainly detects seven short-chain and four medium-chain fatty SP:SAMPLEPREP_SUMMARY acids. Qualitative and quantitative analysis was also performed using internal SP:SAMPLEPREP_SUMMARY standard method. Metabolic extracts were analyzed using the SHIMADZU SP:SAMPLEPREP_SUMMARY GC2030-QP2020 NX gas chromatography-mass spectrometer. The system employed an SP:SAMPLEPREP_SUMMARY HP-FFAP capillary column, and a 1 μL aliquot of the analyte was injected in SP:SAMPLEPREP_SUMMARY split mode (5:1). Helium was used as the carrier gas with a front inlet purge SP:SAMPLEPREP_SUMMARY flow of 3 mL/min and a gas flow rate of 1 mL/min through the column. The initial SP:SAMPLEPREP_SUMMARY temperature was maintained at 50 °C for 1 min, then increased to 150 °C at a SP:SAMPLEPREP_SUMMARY rate of 50 °C/min for 1 min. Subsequently, it was raised to 170 °C at a rate SP:SAMPLEPREP_SUMMARY of 10 °C/min for 1 min, further increased to 210 °C at a rate of 20 °C/min SP:SAMPLEPREP_SUMMARY for 1 min, and finally raised to 240 °C at a rate of 40 °C/min for 1 min. The SP:SAMPLEPREP_SUMMARY injection, transfer line, quad, and ion source temperatures were set at 220 °C, SP:SAMPLEPREP_SUMMARY 240 °C, 150 °C, and 200 °C, respectively. The energy used was -70 eV in SP:SAMPLEPREP_SUMMARY electron impact mode. Mass spectrometry data were acquired in Scan/SIM mode SP:SAMPLEPREP_SUMMARY within the m/z range of 33-150 after a solvent delay of 3 min. Metabolite SP:SAMPLEPREP_SUMMARY identification was performed using an in-house MS database. The pre-processing SP:SAMPLEPREP_SUMMARY of MS raw data involved filtering individual metabolites to retain those with no SP:SAMPLEPREP_SUMMARY more than 50% missing values. Missing values in the original data were simulated SP:SAMPLEPREP_SUMMARY by multiplying the minimum value by a random number between 0.1 and 0.5. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME SHIMADZU GC2030 CH:COLUMN_NAME Agilent HP5-MS (30m x 0.25mm, 0.25 um) CH:SOLVENT_A - CH:SOLVENT_B - CH:FLOW_GRADIENT - CH:FLOW_RATE 350-400 CH:COLUMN_TEMPERATURE 475°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME SHIMADZU MS:INSTRUMENT_TYPE GC2030-QP2020 MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The quantitative results are calculated from the following formula: Calculation MS:MS_COMMENTS formula: C（con）= (Cs∗V1∗V3)/(M∗V2) *1000 C (con) : content of the MS:MS_COMMENTS target compound in the sample, μg/g; Cs: target compound concentration in MS:MS_COMMENTS extract, mg/l; V1: Volume of extract solution added, ML; V2: Take Out pure water MS:MS_COMMENTS supernatant volume, ML; V3: Volume of pure water added, ML; M: weighing sample, MS:MS_COMMENTS MG. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS m/z MS_METABOLITE_DATA_START Samples 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Factors Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Early stage ESCC Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Fator:Normal tissue Acetic acid 16895 80995 255764 10518 117763 10413 5307 34952 19611 9695 22833 7365 7084 4847 11695 5257 11368 42027 20423 26580 6538 10384 8111 34435 9820 16873 26484 17005 13256 14265 28791 11242 Propionic acid 3385 19211 64168 995 3666 1110 703 20656 3560 1017 5613 781 681 778 1214 551 791 6376 1768 2220 705 902 505 12686 1156 1055 1652 710 1002 824 3729 899 Isobutyric acid 6929 104452 88054 1806 75887 2015 1401 17212 14398 1074 1704 1270 2388 578 2819 931 1910 31477 4183 2600 1612 1756 1099 10107 2506 2668 1416 1149 673 1011 5728 1430 Butyric acid 10941 164552 203846 3362 199655 2694 2088 21228 18153 2898 8593 3152 2332 1743 3659 1806 2243 28588 3148 1726 2260 1347 1190 6552 2188 1484 1549 1261 1304 1097 3582 1166 Isovaleric acid 5797 17218 17173 1319 43283 1169 1080 4458 3311 877 1478 866 641 592 967 682 1294 3871 1131 699 1150 725 545 1822 1405 806 668 648 517 505 1662 534 Valeric acid 8977 20915 10773 2280 10343 1892 2116 2596 3558 1894 1568 1469 1157 1184 1105 1089 2100 2817 1399 1188 1642 1177 1030 1112 1838 1252 963 884 1398 963 944 902 Hexanoic acid 15077 14516 5440 6322 8707 10865 11776 16160 31713 34469 21949 14021 16129 7766 3761 8985 3119 11061 10860 5157 13142 13676 5739 3366 14741 6746 3478 2929 15351 3264 3230 5037 Heptanoic acid 963 1062 825 801 1012 1022 1115 1022 1385 1293 1237 877 968 836 729 878 624 949 966 966 992 803 919 1041 868 951 848 818 944 869 789 625 Octanoic acid 6344 5133 10934 7572 13973 18720 15324 8405 6115 6526 6128 9875 9290 8707 8767 5295 12558 7139 8367 5752 15894 13355 15437 12763 12029 7420 8173 16634 14296 12824 10801 7824 Nonanoic acid 11760 15327 13127 12420 14763 9587 12042 12641 12876 19080 19060 16608 17542 13228 13691 10947 11535 14449 13879 14271 12426 12961 19458 15004 15581 13604 11455 15545 11617 14744 10058 17788 Decanoic acid 2943 2484 4657 3467 5535 5084 5763 5058 3029 2989 3038 5929 4328 4665 5733 2234 5381 4138 3258 3842 7305 2799 5922 9201 7135 4006 3715 10139 6929 6949 6483 2744 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CAS molecular formula quantitated m/z HMDB ID KEGG ID PubChem ID Acetic acid 64-19-7 CH3COOH 60.05 HMDB0000042 C00033 176 Propionic acid 1979/9/4 CH3CH2COOH 74.08 HMDB0000237 C00163 1032 Isobutyric acid 79-31-2 C4H8O2 88.11 HMDB0001873 C02632 6590 Butyric acid 107-92-6 C4H8O2 88.11 HMDB0000039 C00246 264 Isovaleric acid 503-74-2 C5H10O2 102.12 HMDB0000718 C08262 10430 Valeric acid 109-52-4 C5H10O2 102.13 HMDB0000892 C00803 7991 Hexanoic acid 142-62-1 C6H12O2 116.16 HMDB0000535 C01585 8892 Heptanoic acid 111-14-8 C7H14O2 130.18 HMDB0000666 C17714 8094 Octanoic acid 124-07-2 C8H16O2 144.21 HMDB0000482 C06423 379 Nonanoic acid 112-05-0 C9H18O2 158.24 HMDB0000847 C01601 8158 Decanoic acid 334-48-5 C10H20O2 172.26 HMDB0000511 C01571 2969 METABOLITES_END #END