#METABOLOMICS WORKBENCH siddia_20220411_153938 DATATRACK_ID:3193 STUDY_ID:ST002233 ANALYSIS_ID:AN003643 PROJECT_ID:PR001423 VERSION 1 CREATED_ON July 24, 2022, 7:29 am #PROJECT PR:PROJECT_TITLE Batch variation of large scale LC-MS metabolomics analysis of human plasma PR:PROJECT_TITLE samples PR:PROJECT_SUMMARY Metabolomics holds the promise to measure and quantify small molecules PR:PROJECT_SUMMARY comprehensively in biological systems, and LC-MS (liquid chromatography coupled PR:PROJECT_SUMMARY mass spectrometry) has become the leading technology in the field. Significant PR:PROJECT_SUMMARY challenges still exist in the computational processing of data from LC-MS PR:PROJECT_SUMMARY metabolomic experiments into metabolite features, including provenance and PR:PROJECT_SUMMARY reproducibility of the current software tools. Current dataset, named HZV029, PR:PROJECT_SUMMARY comprises of 268 data files, from two QC (human pooled plasma) samples that were PR:PROJECT_SUMMARY analyzed repeatedly over 17 batches, on a Thermo Scientific Orbitrap ID-X PR:PROJECT_SUMMARY Tribrid mass spectrometer, coupled with dual liquid chromatography via a PR:PROJECT_SUMMARY Transcend LX-2 System. It provides a unique opportunity to be used as a PR:PROJECT_SUMMARY benchmark dataset to evaluate reproducibility via available choices of different PR:PROJECT_SUMMARY processing tools without knowing the ground truth about these QC samples. PR:INSTITUTE The Jackson Laboratory For Genomic Medicine PR:LABORATORY Shuzhao Li Lab PR:LAST_NAME Siddiqa PR:FIRST_NAME Amnah PR:ADDRESS 10 Discovery Drive , Farmington, CT 06032 PR:EMAIL amnah.siddiqa@jax.org PR:PHONE 404-918 7223 PR:FUNDING_SOURCE 1R01AI149746 (NIH-NIAID) #STUDY ST:STUDY_TITLE Batch variation of large scale LC-MS metabolomics analysis of human plasma ST:STUDY_TITLE samples ST:STUDY_SUMMARY Metabolomics holds the promise to measure and quantify small molecules ST:STUDY_SUMMARY comprehensively in biological systems, and LC-MS (liquid chromatography coupled ST:STUDY_SUMMARY mass spectrometry) has become the leading technology in the field. Significant ST:STUDY_SUMMARY challenges still exist in the computational processing of data from LC-MS ST:STUDY_SUMMARY metabolomic experiments into metabolite features, including provenance and ST:STUDY_SUMMARY reproducibility of the current software tools. Current dataset, named HZV029, ST:STUDY_SUMMARY comprises of 268 data files, from two QC (human pooled plasma) samples that were ST:STUDY_SUMMARY analyzed repeatedly over 17 batches, on a Thermo Scientific Orbitrap ID-X ST:STUDY_SUMMARY Tribrid mass spectrometer, coupled with dual liquid chromatography via a ST:STUDY_SUMMARY Transcend LX-2 System. It provides a unique opportunity to be used as a ST:STUDY_SUMMARY benchmark dataset to evaluate reproducibility via available choices of different ST:STUDY_SUMMARY processing tools without knowing the ground truth about these QC samples. ST:INSTITUTE The Jackson Laboratory For Genomic Medicine ST:LABORATORY Shuzhao Li Lab ST:LAST_NAME Siddiqa ST:FIRST_NAME Amnah ST:ADDRESS 10 Discovery Drive , Farmington, CT 06032 ST:EMAIL amnah.siddiqa@jax.org ST:PHONE 404-918 7223 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 - batch1_MT_20210726_015 SampleType:PooledLiLab Batch=batch1; RAW_FILE_NAME=batch1_MT_20210726_015.raw SUBJECT_SAMPLE_FACTORS - batch1_MT_20210726_059 SampleType:PooledLiLab Batch=batch1; RAW_FILE_NAME=batch1_MT_20210726_059.raw SUBJECT_SAMPLE_FACTORS - batch1_MT_20210726_099 SampleType:PooledLiLab Batch=batch1; RAW_FILE_NAME=batch1_MT_20210726_099.raw SUBJECT_SAMPLE_FACTORS - batch1_MT_20210726_147 SampleType:PooledLiLab Batch=batch1; RAW_FILE_NAME=batch1_MT_20210726_147.raw SUBJECT_SAMPLE_FACTORS - batch1_MT_20210726_189 SampleType:PooledLiLab Batch=batch1; 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RAW_FILE_NAME=batch7_MT_20210801_003K.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_001 SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_001.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_003 SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_003.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_001A SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_001A.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_003C SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_003C.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_087 SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_087.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_089 SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_089.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_001E SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_001E.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_003G SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_003G.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_189 SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_189.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_191 SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_191.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_001I SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_001I.raw SUBJECT_SAMPLE_FACTORS - batch8_MT_20210802_003K SampleType:CommercialQstdPooled Batch=batch8; RAW_FILE_NAME=batch8_MT_20210802_003K.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_001 SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_001.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_003 SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_003.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_001A SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_001A.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_003C SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_003C.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_087 SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_087.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_089 SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_089.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_001E SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_001E.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_003G SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_003G.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_183 SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_183.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_185 SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_185.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_001I SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_001I.raw SUBJECT_SAMPLE_FACTORS - batch9_MT_20210803_003K SampleType:CommercialQstdPooled Batch=batch9; RAW_FILE_NAME=batch9_MT_20210803_003K.raw #COLLECTION CO:COLLECTION_SUMMARY Two different deidentified human plasma pooled samples were used for this study: CO:COLLECTION_SUMMARY a) commercial Qstd plasma pool ; b) biosamples (from 1R01AI149746 (NIH-NIAID) CO:COLLECTION_SUMMARY samples) plasma pool CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY No treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The plasma samples were thawed in ice. Metabolites extraction were carried out SP:SAMPLEPREP_SUMMARY by protein precipitation using extraction solvent, acetonitrile:methanol (8:1, SP:SAMPLEPREP_SUMMARY v/v) containing 0.1% formic acid and isotope labelled Trimethyl-13C3]-caffeine, SP:SAMPLEPREP_SUMMARY [13C5]-L-glutamic acid, [15N2]-Uracil, [15N,13C5]-L-methionine, [13C6]-D-glucose SP:SAMPLEPREP_SUMMARY and [15N]-L-tyrosin as spike-in controls. 30 μl of plasma was taken and 60 μl SP:SAMPLEPREP_SUMMARY of extraction solvent was added. All samples were vortexed and incubated with SP:SAMPLEPREP_SUMMARY shaking at 1000 rpm for 10 min at 4°C followed by centrifugation at 4°C for 15 SP:SAMPLEPREP_SUMMARY min at 15,000 rpm. The supernatant was transferred into mass spec vials and 2 SP:SAMPLEPREP_SUMMARY μl injected into UHPLC-MS. SP:EXTRACTION_METHOD Protein precipitation SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Mobile phase: 10 mM ammonium acetate in 95% acetonitrile containing 0.1% acetic CH:CHROMATOGRAPHY_SUMMARY acid as mobile phase A and 10 mM ammonium acetate in 50% acetonitrile containing CH:CHROMATOGRAPHY_SUMMARY 0.1% acetic acid as mobile phase B were used for HILIC method. Mobile phase CH:CHROMATOGRAPHY_SUMMARY gradient: For HILIC acquisition, following gradient was applied at a flow rate CH:CHROMATOGRAPHY_SUMMARY of 0.55 ml/min: 0-0.1 min: 0% B, 0.10-5.0 min: 98% B, 5.00-5.50 min: 0% B and CH:CHROMATOGRAPHY_SUMMARY 4.5 min for cleaning and equilibration of column.For RP column, following CH:CHROMATOGRAPHY_SUMMARY gradient was applied at a flow rate of 0.4 ml/min: 0-0.1 min: 0% B, 0.10-1.9 CH:CHROMATOGRAPHY_SUMMARY min: 60% B, 1.9-5.0 min: 98% B, 5.00-5.10 min: 0% B and 4.9 min cleaning and CH:CHROMATOGRAPHY_SUMMARY column equilibration. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Scientific Vanquish Horizon UHPLC system CH:COLUMN_NAME HILIC column, AccucoreTM-150-Amide 50 x 2.1 x 2.6 μ CH:COLUMN_TEMPERATURE 45°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS None MS:MS_RESULTS_FILE ST002233_AN003643_Results.txt UNITS:m/z values Has m/z:Yes Has RT:Yes RT units:Seconds #END