#METABOLOMICS WORKBENCH fernandezlab_20210505_151314 DATATRACK_ID:2623 STUDY_ID:ST001778 ANALYSIS_ID:AN002887 PROJECT_ID:000000 VERSION 1 CREATED_ON May 8, 2021, 10:02 am #PROJECT PR:PROJECT_TYPE MS based metabolomics PR:PROJECT_SUMMARY Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap mass PR:PROJECT_SUMMARY spectrometry (MS) are among the highest-performing analytical platforms in PR:PROJECT_SUMMARY metabolomics. Their high mass measurement accuracy and mass resolving power PR:PROJECT_SUMMARY enable detailed investigation of biological metabolomes. Non-targeted MS PR:PROJECT_SUMMARY experiments, however, yield extremely complex datasets that make metabolite PR:PROJECT_SUMMARY annotation very challenging, if not impossible. High-resolution accurate mass PR:PROJECT_SUMMARY measurements greatly facilitate this process by reducing mass errors and PR:PROJECT_SUMMARY spectral overlaps. When applied together with relative isotopic abundance (RIA) PR:PROJECT_SUMMARY measurements, heuristic rules, and constraints during searches, the number of PR:PROJECT_SUMMARY candidate elemental formula(s) can be significantly reduced. Here, we evaluate PR:PROJECT_SUMMARY the performance of two leading analytical MS platforms, Orbitrap ID-X and 12T PR:PROJECT_SUMMARY solariX FT-ICR mass spectrometers, in terms of mass accuracy and RIA PR:PROJECT_SUMMARY measurements, and how these factors affect the assignment of the correct PR:PROJECT_SUMMARY elemental formulae in metabolite annotation. PR:INSTITUTE Georgia Institute of Technology PR:DEPARTMENT Chemistry PR:LABORATORY Fernández PR:FIRST_NAME Danning PR:ADDRESS 901 Atlantic Dr NE, Atlanta, GA, 30332, USA PR:EMAIL dhuang74@gatech.edu PR:PHONE 404-512-7523 PR:PROJECT_TITLE Comparison of High-Resolution Fourier Transform Mass Spectrometry Platforms for PR:PROJECT_TITLE Metabolite Annotation (part II) PR:LAST_NAME Huang #STUDY ST:STUDY_TITLE Comparison of High-Resolution Fourier Transform Mass Spectrometry Platforms for ST:STUDY_TITLE Metabolite Annotation ST:STUDY_SUMMARY Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap mass ST:STUDY_SUMMARY spectrometry (MS) are among the highest-performing analytical platforms in ST:STUDY_SUMMARY metabolomics. Their high mass measurement accuracy and mass resolving power ST:STUDY_SUMMARY enable detailed investigation of biological metabolomes. Non-targeted MS ST:STUDY_SUMMARY experiments, however, yield extremely complex datasets that make metabolite ST:STUDY_SUMMARY annotation very challenging, if not impossible. High-resolution accurate mass ST:STUDY_SUMMARY measurements greatly facilitate this process by reducing mass errors and ST:STUDY_SUMMARY spectral overlaps. When applied together with relative isotopic abundance (RIA) ST:STUDY_SUMMARY measurements, heuristic rules, and constraints during searches, the number of ST:STUDY_SUMMARY candidate elemental formula(s) can be significantly reduced. Here, we evaluate ST:STUDY_SUMMARY the performance of two leading analytical MS platforms, Orbitrap ID-X and 12T ST:STUDY_SUMMARY solariX FT-ICR mass spectrometers, in terms of mass accuracy and RIA ST:STUDY_SUMMARY measurements, and how these factors affect the assignment of the correct ST:STUDY_SUMMARY elemental formulae in metabolite annotation. 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:STUDY_TYPE MS based metabolomics ST:PHONE 4045127523 #SUBJECT SU:SUBJECT_TYPE Synthetic sample #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 - Model mixture 1 Experimental Condition:8M - neg RAW_FILE_NAME=MeOH_8M_neg_1.mzML SUBJECT_SAMPLE_FACTORS - Model mixture 2 Experimental Condition:8M - neg RAW_FILE_NAME=MeOH_8M_neg_2.mzML SUBJECT_SAMPLE_FACTORS - Model mixture 3 Experimental Condition:8M - pos RAW_FILE_NAME=MeOH_8M_pos_1.mzML SUBJECT_SAMPLE_FACTORS - Model mixture 4 Experimental Condition:8M - pos RAW_FILE_NAME=MeOH_8M_pos_2.mzML SUBJECT_SAMPLE_FACTORS - Model mixture 5 Experimental Condition:8M - pos RAW_FILE_NAME=MeOH_8M_pos_3.mzML SUBJECT_SAMPLE_FACTORS - PD1074 C. elegans mixture 1 Experimental Condition:8M - neg RAW_FILE_NAME=Biomatrix_8M_neg_1.mzML SUBJECT_SAMPLE_FACTORS - PD1074 C. elegans mixture 2 Experimental Condition:8M - neg RAW_FILE_NAME=Biomatrix_8M_neg_2.mzML SUBJECT_SAMPLE_FACTORS - PD1074 C. elegans mixture 3 Experimental Condition:8M - neg RAW_FILE_NAME=Biomatrix_8M_neg_3.mzML SUBJECT_SAMPLE_FACTORS - PD1074 C. elegans mixture 4 Experimental Condition:8M - pos RAW_FILE_NAME=Biomatrix_8M_pos_1.mzML SUBJECT_SAMPLE_FACTORS - PD1074 C. elegans mixture 5 Experimental Condition:8M - pos RAW_FILE_NAME=Biomatrix_8M_pos_2.mzML SUBJECT_SAMPLE_FACTORS - PD1074 C. elegans mixture 6 Experimental Condition:8M - pos RAW_FILE_NAME=Biomatrix_8M_pos_3.mzML #COLLECTION CO:COLLECTION_SUMMARY PD1074 C. elegans samples (~10 mg) were collected and lyophilized. CO:SAMPLE_TYPE Worms CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY standard PD1047 C.elegans treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Model Mixture Sample Preparation One hundred and four selected chemical standard SP:SAMPLEPREP_SUMMARY compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used to SP:SAMPLEPREP_SUMMARY prepare stock solutions at a concentration of 0.001 M in LC-MS grade methanol SP:SAMPLEPREP_SUMMARY (Fisher Scientific, Pittsburgh, PA, USA). These standards were selected because SP:SAMPLEPREP_SUMMARY they map to key metabolic pathways, some involved in cancer. If a chemical could SP:SAMPLEPREP_SUMMARY not be completely dissolved in methanol, LC-MS grade water (Fisher Scientific, SP:SAMPLEPREP_SUMMARY Pittsburgh, PA, USA) was added to increase solubility. A pooled sample of 104 SP:SAMPLEPREP_SUMMARY standard compounds was diluted with methanol at a final concentration of 5 μM. SP:SAMPLEPREP_SUMMARY PD1074 C. elegans Bio-mixture Sample Preparation Three 2.0 mm zirconium oxide SP:SAMPLEPREP_SUMMARY beads and ~75 μL volume of 0.5 mm glass beads were added to each lyophilized SP:SAMPLEPREP_SUMMARY PD1074 C. elegans sample (~10 mg). Samples were placed in a TissueLyser II SP:SAMPLEPREP_SUMMARY (QIAGEN, Hilden, Germany) and homogenized at 1,800 rpm, -80 °C for 3 min. One SP:SAMPLEPREP_SUMMARY and a half mL of 80% methanol (in water) was added to each homogenized sample. SP:SAMPLEPREP_SUMMARY Samples were then shaken using an Isotemp high speed shaker (Fisher Scientific, SP:SAMPLEPREP_SUMMARY Pittsburgh, PA, USA) at 1,500 rpm for 30 min, and centrifuged at 22,100 g for 5 SP:SAMPLEPREP_SUMMARY min. The supernatant was collected, dried and stored at -80 °C. Prior to MS SP:SAMPLEPREP_SUMMARY analysis, dried C. elegans matrices were resuspended with 1 mL LC-MS grade SP:SAMPLEPREP_SUMMARY methanol containing the 104 standard compounds at a 5 μM concentration. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME none CH:COLUMN_NAME none #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Solarix FT-ICR-MS MS:INSTRUMENT_TYPE FT-ICR MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Samples were analyzed in positive and negative ion modes with direct infusion MS:MS_COMMENTS FT-ICR MS. A spectral size of 8 M was used for data acquisition with an ion MS:MS_COMMENTS accumulation time of 0.025 s and a scan range of 73.71 – 1000.00 m/z. Average MS:MS_COMMENTS scans were set to 200. Raw data files were converted into mzML format using MS:MS_COMMENTS MSConvert. Spectral features were extracted using MZmine 2.51. MS:CAPILLARY_VOLTAGE -4.5kV MS:DRY_GAS_FLOW 4.0 L/min MS:DRY_GAS_TEMP 180 °C MS:NEBULIZER 0.5bar #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Da MS_METABOLITE_DATA_START Samples Model mixture 1 Model mixture 2 PD1074 C. elegans mixture 1 PD1074 C. elegans mixture 2 PD1074 C. elegans mixture 3 Factors Experimental Condition:8M - neg Experimental Condition:8M - neg Experimental Condition:8M - neg Experimental Condition:8M - neg Experimental Condition:8M - neg glycolic acid 75.00876 75.00877 75.00877 75.00877 75.00877 Pyruvate 87.00878 87.00879 87.00879 87.00879 87.00879 Alanine n/a n/a n/a n/a n/a L-Lactate 89.02441 89.02441 89.02443 89.02443 89.02443 Acetoacetate 101.02441 101.02441 101.02443 101.02443 101.02443 2-Aminoisobutyric acid 102.05606 102.05606 102.05607 102.05607 102.05608 Choline n/a n/a n/a n/a n/a Malonic acid 103.00368 103.00368 103.00368 103.00369 103.00369 L-Serine 104.03532 104.03532 104.03533 104.03534 104.03534 L-Glyceric acid 105.01934 105.01935 105.01936 105.01936 105.01936 Cytosine n/a n/a n/a n/a n/a Histamine n/a n/a n/a n/a n/a Uracil 111.02 111.02 111.02002 111.02002 111.02002 L-Proline (Reagents Plus) n/a n/a n/a n/a n/a Fumarate dibasic 115.0037 115.0037 115.00372 115.00372 115.00372 N-Acetylglycine 116.03532 116.03532 116.03533 116.03533 116.03533 L-Valine 116.07172 116.07172 116.07173 116.07173 116.07173 Succinate (dibasic) 117.01933 117.01933 117.01935 117.01935 117.01935 L-Threonine 118.05097 118.05097 118.05099 118.05099 118.05099 D-(-)-Erythrose 119.03499 119.03499 119.035 119.035 119.035 Nicotinamide n/a n/a n/a n/a n/a Nicotinic acid 122.02475 122.02475 122.02477 122.02477 122.02477 Taurine 124.0074 124.0074 124.00742 124.00742 124.00742 Pyroglutamic acid 128.03534 128.03534 128.03535 128.03535 128.03535 Citraconic acid 129.01935 129.01935 129.01936 129.01936 129.01936 trans-4-Hydroxy-L-proline 130.05098 130.05098 130.05099 130.05099 130.05099 creatine 130.06221 130.06221 130.06223 130.06223 130.06223 L-Leucine 130.08737 130.08737 130.08739 130.08739 130.08739 Glutaric acid 131.035 131.035 131.035 131.035 131.03502 L-Asparagine 131.04623 131.04623 131.04625 131.04625 131.04625 L-Leucic acid 131.0714 131.0714 131.0714 131.0714 131.07141 L-Ornithine 131.08261 131.08261 131.08263 131.08263 131.08263 L-Aspartic acid 132.03024 132.03026 132.03026 132.03026 132.03027 DL-Malic acid 133.01425 133.01425 133.01427 133.01427 133.01427 Adenine 134.04724 134.04724 134.04726 134.04726 134.04726 Hypoxanthine 135.03125 135.03125 135.03127 135.03127 135.03127 Anthranilic acid 136.04042 136.04044 136.04044 136.04044 136.04045 Acetylcholine n/a n/a n/a n/a n/a alpha-Ketoglutaric acid 145.01425 145.01427 145.01427 145.01427 145.01427 Adipic acid 145.05066 145.05066 145.05066 145.05066 145.05066 L-Glutamine 145.06189 145.06189 145.06189 145.06189 145.0619 L-Lysine n/a n/a n/a n/a n/a L-Glutamic acid n/a n/a 146.04591 146.04591 146.04591 L-Methionine 148.04378 148.04379 148.04379 148.04379 148.04379 Xanthine 151.02615 151.02617 151.02617 151.02617 151.02617 Cystamine n/a n/a n/a n/a n/a Histidine 154.06223 154.06223 154.06224 154.06224 154.06224 Orotic acid 155.00983 155.00984 155.00986 155.00986 155.00986 Allantoin 157.03671 157.03673 157.03673 157.03673 157.03673 DL-2-Aminoadipic acid 160.06155 160.06155 160.06157 160.06157 160.06157 Carnitine n/a n/a n/a n/a n/a Phenylpyruvic acid 163.04008 163.04008 163.0401 163.0401 163.0401 7-methylguanine 164.0578 164.0578 164.0578 164.05782 164.05782 L-Phenylalanine 164.07173 164.07173 164.07175 164.07175 164.07175 Phospho(enol)pyruvic acid 166.97513 166.97513 166.97513 166.97513 166.97514 Uric Acid 167.02107 167.02109 167.02109 167.02109 167.02109 trans-Aconitic acid 173.00916 173.00917 173.00917 173.00919 173.00919 Shikimic acid 173.04555 173.04555 173.04556 173.04556 173.04556 Arginine 173.1044 173.1044 173.10442 173.1044 173.10443 L-citrulline 174.08842 174.08844 174.08844 174.08844 174.08846 Hippuric acid 178.05098 178.05098 178.05098 178.05098 178.05098 4-Hydroxyphenylpyruvic acid 179.03499 179.035 179.035 179.035 179.035 myo-Inositol 179.05614 179.05614 179.05612 179.05614 179.05612 L-Tyrosine 180.06665 180.06665 180.06665 180.06665 180.06667 Homovanillic acid 181.05066 181.05066 181.05067 181.05067 181.05067 D-Sorbitol 181.07178 181.07179 181.07181 181.07181 181.07181 4-Pyridoxic acid 182.04588 182.04588 182.0459 182.0459 182.0459 DL-Normetanephrine 182.08226 182.08228 182.08231 182.08229 182.08237 Kynurenic acid 188.03534 188.03534 188.03535 188.03535 188.03535 D-Glucuronic acid 193.03542 193.03542 193.03542 193.03542 193.03543 L-Tryptophan 203.0826 203.0826 203.0826 203.0826 203.08261 Xanthurenic acid 204.03024 204.03024 204.03026 204.03026 204.03026 L-Kynurenine 207.07755 207.07755 207.07755 207.07755 207.07755 undecanedioic acid 215.12888 215.12889 215.12889 215.12889 215.12889 5'-Hydroxy-L-tryptophan 219.07755 219.07755 219.07755 219.07755 219.07755 3-Nitro-L-Tyrosine 225.05171 225.05171 225.05171 225.05171 225.05171 2'-Deoxyuridine 227.06735 227.06737 227.06737 227.06737 227.06737 Myristic acid 227.20168 227.20168 227.20168 227.20169 227.20169 Melatonin 231.11391 231.11392 231.11391 231.11392 231.11392 L-Cystine 239.01662 239.01662 239.01665 239.01663 239.01662 Cytidine 242.07823 242.07825 242.07822 242.07823 242.07823 Uridine 243.06229 243.0623 243.06229 243.06229 243.06229 Biotin 243.08092 243.08092 243.0809 243.0809 243.0809 Pyridoxal 5' phosphate 246.01732 246.01732 246.0173 246.0173 246.01732 α-D-Glucose 1-phosphate 259.02246 259.02246 259.02246 259.02246 259.02246 O-phospho-L-tyrosine 260.03296 260.03296 260.03293 260.03293 260.03293 Adenosine 266.08948 266.08948 266.08945 266.08945 266.08945 Inosine 267.07352 267.07352 267.07349 267.07349 267.07349 Heptadecanoic acid 269.24866 269.24866 269.24863 269.24863 269.24866 Guanosine 282.08444 282.08444 282.08441 282.08441 282.08441 Xanthosine 283.06845 283.06845 283.06842 283.06842 283.06842 2'-Deoxycytidine 5'-monophosphate 306.04968 306.04971 306.04968 306.04968 306.04968 L-Glutathione Reduced 306.07648 306.07651 306.07648 306.07648 306.07648 Cytidine 5'-Monophosphate 322.04468 322.04468 322.04459 322.04459 322.04459 D-Fructose 1,6-biphosphate 338.98877 338.98877 338.98871 338.98871 338.98871 Sucrose 341.10892 341.10892 341.10886 341.10886 341.10886 Gibberellic acid 345.13434 345.13434 345.13425 345.13425 345.13425 Adenosine 5'-Monophosphate 346.05582 346.05582 346.05576 346.05576 346.05576 Inosine 5'-monophosphate 347.03983 347.03983 347.03976 347.03976 347.03976 Chenodeoxycholate 391.28543 391.28543 391.28534 391.28534 391.28537 Glycochenodeoxycholate 448.3067 448.3067 448.30664 448.30664 448.30664 Glycocholic acid 464.30185 464.30188 464.30164 464.30164 464.30164 Adenosine 5'-triphosphate 505.98862 505.98865 505.98843 505.98843 505.98846 L-Glutathione Oxidized 611.14478 611.14484 611.14459 611.14459 611.14459 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Elemental Formula Theoretical Mass (Neg) glycolic acid C2H4O3 75.00877 Pyruvate C3H4O3 87.00877 Alanine C3H7NO2 88.0404 L-Lactate C3H6O3 89.02442 Acetoacetate C4H6O3 101.02442 2-Aminoisobutyric acid C4H9NO2 102.05605 Choline C5H14NO 102.09244 Malonic acid C3H4O4 103.00368 L-Serine C3H7NO3 104.03532 L-Glyceric acid C3H6O4 105.01933 Cytosine C4H5N3O 110.03599 Histamine C5H9N3 110.07237 Uracil C4H4N2O2 111.02 L-Proline (Reagents Plus) C5H9NO2 114.0555 Fumarate dibasic C4H4O4 115.00368 N-Acetylglycine C4H7NO3 116.03532 L-Valine C5H11NO2 116.0717 Succinate (dibasic) C4H6O4 117.01933 L-Threonine C4H9NO3 118.05097 D-(-)-Erythrose C4H8O4 119.03498 Nicotinamide C6H6N2O 121.04074 Nicotinic acid C6H5NO2 122.02475 Taurine C2H7NO3S 124.00739 Pyroglutamic acid C5H7NO3 128.03532 Citraconic acid C5H6O4 129.01933 trans-4-Hydroxy-L-proline C5H9NO3 130.05097 creatine C4H9N3O2 130.0622 L-Leucine C6H13NO2 130.08735 Glutaric acid C5H8O4 131.03498 L-Asparagine C4H8N2O3 131.04622 L-Leucic acid C6H12O3 131.07137 L-Ornithine C5H12N2O2 131.0826 L-Aspartic acid C4H7NO4 132.03023 DL-Malic acid C4H6O5 133.01425 Adenine C5H5N5 134.04722 Hypoxanthine C5H4N4O 135.03123 Anthranilic acid C7H7NO2 136.0404 Acetylcholine C7H16NO2 144.103 alpha-Ketoglutaric acid C5H6O5 145.01425 Adipic acid C6H10O4 145.05063 L-Glutamine C5H10N2O3 145.06187 L-Lysine C6H14N2O2 145.0977 L-Glutamic acid C5H9NO4 146.04588 L-Methionine C5H11NO2S 148.04377 Xanthine C5H4N4O2 151.02615 Cystamine C4H12N2S2 151.03691 Histidine C6H9N3O2 154.0622 Orotic acid C5H4N2O4 155.00983 Allantoin C4H6N4O3 157.03671 DL-2-Aminoadipic acid C6H11NO4 160.06153 Carnitine C7H15NO3 160.09792 Phenylpyruvic acid C9H8O3 163.04007 7-methylguanine C6H7N5O 164.05778 L-Phenylalanine C9H11NO2 164.0717 Phospho(enol)pyruvic acid C3H5O6 166.9751 Uric Acid C5H4N4O3 167.02106 trans-Aconitic acid C6H6O6 173.00916 Shikimic acid C7H10O5 173.04555 Arginine C6H14N4O2 173.1044 L-citrulline C6H13N3O3 174.08841 Hippuric acid C9H9NO3 178.05097 4-Hydroxyphenylpyruvic acid C9H8O4 179.03498 myo-Inositol C6H12O6 179.05611 L-Tyrosine C9H11NO3 180.06662 Homovanillic acid C9H10O4 181.05063 D-Sorbitol C6H14O6 181.07176 4-Pyridoxic acid C8H9NO4 182.04588 DL-Normetanephrine C9H13NO3 182.08227 Kynurenic acid C10H7NO3 188.03532 D-Glucuronic acid C6H10O7 193.03538 L-Tryptophan C11H12N2O2 203.0826 Xanthurenic acid C10H7NO4 204.03023 L-Kynurenine C10H12N2O3 207.07752 undecanedioic acid C11H20O4 215.12888 5'-Hydroxy-L-tryptophan C11H12N2O3 219.07752 3-Nitro-L-Tyrosine C9H10N2O5 225.0517 2'-Deoxyuridine C9H12N2O5 227.06735 Myristic acid C14H28O2 227.20165 Melatonin C13H16N2O2 231.1139 L-Cystine C6H12N2O4S 239.01657 Cytidine C9H13N3O5 242.07824 Uridine C9H12N2O6 243.06226 Biotin C10H16N2O3S 243.08089 Pyridoxal 5' phosphate C8H10NO6P 246.0173 α-D-Glucose 1-phosphate C6H13O9P 259.02244 O-phospho-L-tyrosine C9H12NO6P 260.03295 Adenosine C10H13N5O4 266.08948 Inosine C10H12N4O5 267.07349 Heptadecanoic acid C17H34O2 269.2486 Guanosine C10H13N5O5 282.08439 Xanthosine C10H12N4O6 283.06841 2'-Deoxycytidine 5'-monophosphate C9H14N3O7P 306.04966 L-Glutathione Reduced C10H17N3O6S 306.07653 Cytidine 5'-Monophosphate C9H14N3O8P 322.04457 D-Fructose 1,6-biphosphate C6H14O12P2 338.98877 Sucrose C12H22O11 341.10894 Gibberellic acid C19H22O6 345.13436 Adenosine 5'-Monophosphate C10H14N5O7P 346.05581 Inosine 5'-monophosphate C10H13N4O8P 347.03982 Chenodeoxycholate C24H40O4 391.28538 Glycochenodeoxycholate C26H43NO5 448.30685 Glycocholic acid C26H43NO6 464.30176 Adenosine 5'-triphosphate C10H16N5O13P3 505.98847 L-Glutathione Oxidized C20H32N6O12S2 611.14469 METABOLITES_END #END