#METABOLOMICS WORKBENCH Metadata_Spleen DATATRACK_ID:195 STUDY_ID:ST000133 ANALYSIS_ID:AN000215 VERSION 1 CREATED_ON 08-08-2023 #PROJECT PR:PROJECT_TITLE 1H NMR Metabolomics Study of Metastatic Melanoma in C57BL/6J Mouse Spleen PR:INSTITUTE Pacific Northwest National Laboratory PR:DEPARTMENT Fundamental & Computational Sciences PR:LAST_NAME Hu PR:FIRST_NAME Jianzhi PR:EMAIL jianzhi.hu@pnnl.gov PR:DOI http://dx.doi.org/10.21228/M8B594 #STUDY ST:STUDY_TITLE 1H NMR Metabolomics Study of Metastatic Melanoma in C57BL/6J Mouse Spleen ST:STUDY_TYPE Tissue Extracts Comparison ST:STUDY_SUMMARY Tissue extracts from metastatic melanoma mouse spleen and controls were compared ST:STUDY_SUMMARY via NMR based metabolomic analysis ST:INSTITUTE Pacific Northwest National Laboratory ST:DEPARTMENT Fundamental & Computational Sciences ST:LAST_NAME Hu ST:FIRST_NAME Jianzhi ST:EMAIL jianzhi.hu@pnnl.gov ST:SUBMIT_DATE 2015-01-08 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:SPECIES_GROUP Mammal #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 7A10 Disease State:No SUBJECT_SAMPLE_FACTORS - 7B10 Disease State:No SUBJECT_SAMPLE_FACTORS - 7C10 Disease State:No SUBJECT_SAMPLE_FACTORS - 7D10 Disease State:No SUBJECT_SAMPLE_FACTORS - 7E 10 Disease State:No SUBJECT_SAMPLE_FACTORS - 6A10 Disease State:Yes SUBJECT_SAMPLE_FACTORS - 6B10 Disease State:Yes SUBJECT_SAMPLE_FACTORS - 6C10 Disease State:Yes SUBJECT_SAMPLE_FACTORS - 6D10 Disease State:Yes SUBJECT_SAMPLE_FACTORS - 6E 10 Disease State:Yes #COLLECTION CO:COLLECTION_SUMMARY Spleens were dissected and snap-frozen at -80°C before NMR spectroscopy and CO:COLLECTION_SUMMARY morphology analysis. CO:SAMPLE_TYPE Tissue #TREATMENT TR:TREATMENT_SUMMARY A total of 12 six-week-old C57BL/6J male mice at the age of week-7 the mice were TR:TREATMENT_SUMMARY randomly divided into two groups, an experimental group (n=7) that received TR:TREATMENT_SUMMARY subcutaneous injection with suspended 105 B16-F10 tumor cells at four flank TR:TREATMENT_SUMMARY locations on each leg, and a control group (n=5) that were injected with the TR:TREATMENT_SUMMARY same amount of PBS at same places. The animals were fed a standard diet and TR:TREATMENT_SUMMARY housed one mouse per cage and maintained in a temperature-controlled room TR:TREATMENT_SUMMARY (22-25°C, 45% humidity) on a 12:12h dark-light cycle. All mice, each of which TR:TREATMENT_SUMMARY was weighed weekly, were allowed free access to water and food before being TR:TREATMENT_SUMMARY sacrificed by CO2 asphyxiation at the age of fourteen weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cold Methanol/H2O/Chloroform Extraction SP:EXTRACTION_METHOD Step 1: The pre-weighted intact frozen tissue was homogenized in ice bath after SP:EXTRACTION_METHOD adding 0.25 ml MeOH and 0.053 ml H2O for each sample containing 30 to 60 mg of SP:EXTRACTION_METHOD tissue, followed by vortexing the mixture and then adding 0.125 ml chloroform, SP:EXTRACTION_METHOD vortexing again. Step 2: 0.125 ml chloroform and 0.125 ml water was added to SP:EXTRACTION_METHOD the sample and then vortexed again, followed by transferring different layers SP:EXTRACTION_METHOD into glass vials separately with syringes after the mixture being left on ice SP:EXTRACTION_METHOD for 15 min and centrifuged at 8000rpm for 2 min at 4°C. Finally, the solvents SP:EXTRACTION_METHOD were removed by employing lyophilizer (MeOH/H2O layer, hydrophilic metabolites) SP:EXTRACTION_METHOD or by evaporating under dry nitrogen gas (CHCl3 layer, hydrophobic metabolites). SP:EXTRACTION_METHOD The extracts were then stored at -80°C before performing NMR measurements. #CHROMATOGRAPHY #ANALYSIS AN:ANALYSIS_TYPE NMR AN:SOFTWARE_VERSION Vnmrj/ 3.2; Chenomx/7.6 #NMR NM:INSTRUMENT_NAME Varian NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D 1H NM:FIELD_FREQUENCY_LOCK Deuterium NM:STANDARD_CONCENTRATION 0.5 mM NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_PROBE 5mm HCN probe with Z axis-gradient NM:NMR_SOLVENT D2O/CDCl3 NM:NMR_TUBE_SIZE 5mm * 8 in NM:SHIMMING_METHOD Gradient shim NM:PULSE_SEQUENCE PRESAT NM:WATER_SUPPRESSION presat NM:PULSE_WIDTH 6.5 us NM:RECEIVER_GAIN 60 NM:CHEMICAL_SHIFT_REF_CPD TSP-d4 NM:TEMPERATURE 293 K NM:NUMBER_OF_SCANS 8192 NM:DUMMY_SCANS 2 NM:ACQUISITION_TIME 1.9137 s NM:RELAXATION_DELAY 2 NM:SPECTRAL_WIDTH 10000 Hz NM:NUM_DATA_POINTS_ACQUIRED 38274 NM:LINE_BROADENING 0.5 #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS normalized conc. (uM/mg) NMR_METABOLITE_DATA_START Samples 7A10 7B10 7C10 7D10 7E 10 6A10 6B10 6C10 6D10 6E 10 Factors Disease State:No Disease State:No Disease State:No Disease State:No Disease State:No Disease State:Yes Disease State:Yes Disease State:Yes Disease State:Yes Disease State:Yes 2-Aminobutyrate 0.8000 0.4957 0.6266 0.7410 0.8453 0.7936 1.0283 1.2403 0.9526 1.7103 2^-Deoxyguanosine 0.0098 0.0086 0.0077 0.0180 0.0166 0.0560 0.0256 0.0389 0.0368 0.0397 2-Oxoglutarate 0.4918 0.3707 0.3120 0.3417 0.3923 0.4544 0.4265 1.2367 0.9579 0.4802 3-Hydroxybutyrate 0.1574 0.2457 0.1765 0.1942 0.2127 0.1408 0.1188 0.2085 0.1526 0.2421 3-Hydroxyisobutyrate 0.0525 0.1164 0.0332 0.0468 0.0718 0.0464 0.0715 0.0989 0.0763 0.0675 Acetate 0.9082 2.1034 0.9309 1.2230 1.1713 0.6320 2.2942 1.1484 1.2316 1.4365 Adenosine 0.0164 0.0129 0.0358 0.0144 0.0055 0.0192 0.0162 0.0247 0.0316 0.0238 ADP 0.0328 0.0517 0.0767 0.0612 0.0580 0.0736 0.0621 0.0742 0.0263 0.0198 Alanine 7.0033 6.9698 4.3964 7.0360 5.6796 8.2304 9.5331 13.2792 11.2658 14.6111 Alloisoleucine 1.3934 1.9353 1.3708 2.2698 2.1713 1.2608 1.7449 3.2968 2.0000 3.4563 AMP 0.0197 0.0043 0.0102 0.0072 0.0193 0.0576 0.0499 0.0283 0.0289 0.0278 Arginine 3.8721 3.4052 2.6573 3.6223 4.5552 2.5968 2.1633 5.0989 4.8921 6.4563 Asparagine 2.0230 2.0129 1.2864 2.0180 1.5083 2.2320 2.5047 4.0989 4.1368 4.6667 Aspartate 10.9016 9.4655 7.8849 10.2482 10.2597 6.2272 6.1228 7.3781 6.7105 8.0198 ATP 0.1607 0.1336 0.1739 0.1799 0.1630 0.0960 0.0742 0.0530 0.0974 0.0913 Benzoate 0.0557 0.0517 0.0281 0.0540 0.0414 0.0288 0.0243 0.0777 0.0526 0.0278 Betaine 0.4525 0.1983 0.2379 0.3849 0.3122 0.6464 0.5560 0.2756 0.2842 0.8532 Choline 1.6787 1.0819 0.9668 1.1619 1.0497 1.2944 0.7112 2.0424 1.7763 4.0595 Citrate 0.3803 0.4741 0.3223 0.4173 0.4945 0.7840 0.6140 0.5053 0.4500 0.5079 Creatine 0.5475 0.3276 0.3197 0.5036 0.4669 0.2592 0.2186 0.4276 0.2684 0.4127 Creatine phosphate 0.4131 0.5302 0.2199 0.1978 0.1492 0.3200 0.2767 0.7774 0.6974 0.9246 Creatinine 0.6787 0.5776 0.5550 0.5108 0.4696 0.4208 0.1242 0.5300 0.3974 0.5397 Cytidine 0.7508 0.6552 0.3325 0.4281 0.3039 0.0848 0.0715 0.6042 0.2684 0.8135 dCTP 0.0787 0.0603 0.0384 0.0612 0.0663 0.1264 0.1336 0.1979 0.1211 0.1905 DSS-d6 (Chemical Shape Indicator) 16.3738 21.5603 12.8056 17.9748 13.8011 8.0032 6.7476 17.6926 13.1526 19.8214 dTTP 0.0262 0.0216 0.0307 0.0144 0.0166 0.0320 0.0189 0.0212 0.0211 0.0357 Ethanol 5.5213 2.8793 0.9003 1.5935 2.4199 3.0016 2.5641 3.9117 1.0921 3.9127 Ethanolamine 1.4557 1.2543 0.7801 1.2518 1.0110 0.8048 0.6788 1.6325 1.5605 2.5317 Formate 1.0459 0.9871 0.4476 1.0755 0.6188 0.4832 0.1984 0.9293 0.6658 0.7540 Fucose 0.2197 0.2759 0.2302 0.2410 0.2569 0.2144 0.1808 0.4028 0.4579 0.1190 Fumarate 0.0885 0.0948 0.0895 0.0899 0.0608 0.0944 0.1323 0.0883 0.0947 0.1270 Glucose 6.8557 3.9871 4.6138 4.6942 4.1298 4.7104 5.2321 6.4240 4.4158 5.2262 Glutamate 14.0393 14.7500 11.5806 14.7374 13.5083 8.8592 8.1903 9.8975 9.9237 10.4167 Glutamine 5.2557 4.1767 3.4220 5.0899 4.9144 3.2928 3.5682 6.5018 5.8000 7.7500 Glutathione 1.2623 1.6164 1.8056 1.8381 1.7348 3.0384 3.1215 2.1802 2.3079 1.8571 Glycerol 1.0557 0.7672 1.7494 0.8201 1.5221 0.4560 0.3846 0.8304 0.9342 1.9563 Glycine 8.2525 8.2974 4.8465 7.4137 5.2182 8.3936 9.4804 14.3428 12.9342 15.2579 GTP 0.0164 0.0129 0.0153 0.0108 0.0166 0.0336 0.0283 0.0424 0.0447 0.0437 Histamine 0.1016 0.1293 0.1355 0.1439 0.1050 0.2400 0.2024 0.2332 0.1868 0.2262 Histidine 0.4721 0.4957 0.2864 0.4424 0.2818 0.3312 0.3941 0.9258 0.8737 1.2460 Hypoxanthine 1.3672 1.8448 1.3018 1.5791 1.1851 1.4960 1.1417 1.8799 1.7158 1.0556 Inosine 0.2098 0.2845 0.9923 0.5683 1.0691 0.8096 0.8974 0.3675 0.1763 0.2341 Isobutyrate 1.1934 1.3491 0.8133 1.3993 1.0994 0.9904 1.1255 2.8869 2.2132 3.0317 Isocitrate 4.4328 3.1379 4.5882 5.2842 4.7431 3.2656 1.7233 4.0601 3.9553 4.5317 Isoleucine 2.8689 3.2069 1.9923 3.3129 2.5801 2.2352 2.6397 6.6290 5.1605 7.0159 Lactate 12.6426 12.5862 10.6189 18.1511 18.1215 17.5152 17.1120 13.5336 17.0421 19.9960 Leucine 1.1016 1.3448 0.8926 1.2446 0.7900 1.3744 1.2591 2.6148 2.4053 2.2183 Lysine 3.3246 3.9957 2.6215 3.0108 2.2652 2.4144 3.2794 6.6714 6.0526 7.3889 Malate 1.7082 1.4310 1.7596 1.4460 1.5000 2.9440 2.4899 2.3640 2.4289 2.1389 Methanol 27.7934 11.9009 16.8210 20.6871 16.4972 39.4432 13.0769 22.3039 13.6658 19.7857 Methionine 1.3377 1.6767 1.0818 1.7698 1.3122 1.1504 1.3779 2.4523 2.2658 2.8016 myo-Inositol 4.1344 3.5690 3.3427 5.0072 3.7376 1.6304 1.5709 5.1166 4.8158 5.7540 Niacinamide 0.4000 0.3621 0.3811 0.4173 0.3729 0.3232 0.2726 0.3110 0.2921 0.3175 O-Phosphocholine 0.6689 1.0043 0.3939 0.5827 0.4392 0.6176 0.3279 1.1661 0.7526 0.8175 O-Phosphoethanolamine 10.1541 8.6336 7.9386 9.4892 9.6934 3.9568 5.3036 5.3534 4.8447 4.3770 Oxypurinol 11.6393 8.8621 8.5396 9.0252 6.7873 9.2048 7.7638 9.0212 9.4763 9.1508 Phenylalanine 1.3541 1.4267 0.9693 1.5144 1.1298 1.1008 1.4345 2.9152 2.3158 3.1587 pi-Methylhistidine 0.0820 0.0345 0.0895 0.0827 0.0442 0.1168 0.2240 0.1979 0.1395 0.1032 Serine 4.3115 3.3793 2.0639 3.0468 3.3702 2.4528 3.9703 8.8799 8.2316 12.8100 sn-Glycero-3-phosphocholine 5.1148 4.1034 4.0767 4.9784 4.7348 1.9040 1.3914 6.9117 2.1000 10.0675 Taurine 42.7410 35.1940 34.2302 40.2914 41.8785 25.5472 24.4696 29.4028 29.8947 31.2579 Threonine 1.8525 3.0431 1.6368 2.5827 2.6215 2.8800 2.8138 6.6148 4.0763 3.9683 Thymidine 0.0393 0.0560 0.0179 0.0324 0.0304 0.0864 0.0621 0.1166 0.0763 0.1310 Trimethylamine 0.0164 0.0216 0.0077 0.0108 0.0110 0.0192 0.0162 0.0530 0.0316 0.0516 Trimethylamine N-oxide 0.3016 0.1638 0.4271 0.6475 1.0718 0.2896 0.4534 1.2686 0.2605 1.0675 Tryptophan 0.2525 0.3060 0.2251 0.3022 0.2182 0.2032 0.1957 0.5618 0.4500 0.5952 Tyramine 0.1738 0.1509 0.0946 0.1583 0.2072 0.1696 0.0985 0.1166 0.3868 0.3333 Tyrosine 2.0066 2.1034 1.2353 1.8381 1.2762 1.0880 1.5101 3.1908 2.7579 3.9921 UDP-galactose 0.0525 0.0603 0.2430 0.0540 0.0856 0.1424 0.1039 0.0742 0.1816 0.0675 UDP-glucuronate 0.0426 0.0388 0.1969 0.0432 0.1188 0.2160 0.1242 0.0777 0.0421 0.0556 Uracil 0.5082 0.4224 0.3197 0.3669 0.2238 0.3856 0.3063 1.3428 1.0395 1.3968 Uridine 1.0689 0.9397 0.8133 0.9245 0.7735 0.9552 0.9744 0.9859 1.1684 0.7540 Valine 1.5443 1.7241 1.0793 1.8237 1.4448 1.2128 1.3765 3.4417 2.5842 3.7580 Xanthine 0.0820 0.0905 0.1458 0.0647 0.0387 0.3936 0.3455 0.4311 0.6289 0.5119 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant 2-Aminobutyrate 6657 PNNL_Jianzhi_ID 2'-Deoxyguanosine 187790 C00330 PNNL_Jianzhi_ID 2-Oxoglutarate 51 C00026 PNNL_Jianzhi_ID 3-Hydroxybutyrate 441 C01089 PNNL_Jianzhi_ID 3-Hydroxyisobutyrate 87 C01188 PNNL_Jianzhi_ID Acetate 176 C00033 PNNL_Jianzhi_ID Adenosine 60961 C00212 PNNL_Jianzhi_ID ADP 6022 C00008 PNNL_Jianzhi_ID Alanine 5950 C00041 PNNL_Jianzhi_ID Alloisoleucine 99288 PNNL_Jianzhi_ID AMP 6083 C00020 PNNL_Jianzhi_ID Arginine 6322 C00062 PNNL_Jianzhi_ID Asparagine 6267 C00152 PNNL_Jianzhi_ID Aspartate 5960 C00049 PNNL_Jianzhi_ID ATP 5957 C00002 PNNL_Jianzhi_ID Benzoate 243 C00180 PNNL_Jianzhi_ID Betaine 247 C00719 PNNL_Jianzhi_ID Choline 305 C00114 PNNL_Jianzhi_ID Citrate 311 C00158 PNNL_Jianzhi_ID Creatine 586 C00300 PNNL_Jianzhi_ID Creatine phosphate 587 C02305 PNNL_Jianzhi_ID Creatinine 588 C00791 PNNL_Jianzhi_ID Cytidine 6175 C00475 PNNL_Jianzhi_ID dCTP 65091 C00458 PNNL_Jianzhi_ID DSS-d6 (Chemical Shape Indicator) PNNL_Jianzhi_ID dTTP 64968 C00459 PNNL_Jianzhi_ID Ethanol 702 C00469 PNNL_Jianzhi_ID Ethanolamine 700 C00189 PNNL_Jianzhi_ID Formate 284 C00058 PNNL_Jianzhi_ID Fucose 19466 C01019 PNNL_Jianzhi_ID Fumarate 444972 C00122 PNNL_Jianzhi_ID Glucose 5793 C00031 PNNL_Jianzhi_ID Glutamate PNNL_Jianzhi_ID Glutamine 5961 C00064 PNNL_Jianzhi_ID Glutathione 124886 C00051 PNNL_Jianzhi_ID Glycerol 753 C00116 PNNL_Jianzhi_ID Glycine 750 C00037 PNNL_Jianzhi_ID GTP 6830 C00044 PNNL_Jianzhi_ID Histamine 774 C00388 PNNL_Jianzhi_ID Histidine 6274 C00135 PNNL_Jianzhi_ID Hypoxanthine 790 C00262 PNNL_Jianzhi_ID Inosine 6021 C00294 PNNL_Jianzhi_ID Isobutyrate 6590 C02632 PNNL_Jianzhi_ID Isocitrate 1198 C00311 PNNL_Jianzhi_ID Isoleucine 6306 C00407 PNNL_Jianzhi_ID Lactate 107689 C00186 PNNL_Jianzhi_ID Leucine 6106 C00123 PNNL_Jianzhi_ID Lysine 5962 C00047 PNNL_Jianzhi_ID Malate 222656 C00149 PNNL_Jianzhi_ID Methanol 887 C00132 PNNL_Jianzhi_ID Methionine 6137 C00073 PNNL_Jianzhi_ID myo-Inositol 892 C00137 PNNL_Jianzhi_ID Niacinamide 936 C00153 PNNL_Jianzhi_ID O-Phosphocholine 135437 PNNL_Jianzhi_ID O-Phosphoethanolamine 1015 C00346 PNNL_Jianzhi_ID Oxypurinol 4644 C07599 PNNL_Jianzhi_ID Phenylalanine 6140 C00079 PNNL_Jianzhi_ID pi-Methylhistidine 92105 C01152 PNNL_Jianzhi_ID Serine 5951 C00065 PNNL_Jianzhi_ID sn-Glycero-3-phosphocholine 657272 PNNL_Jianzhi_ID Taurine 1123 C00245 PNNL_Jianzhi_ID Threonine 6288 C00188 PNNL_Jianzhi_ID Thymidine 5789 C00214 PNNL_Jianzhi_ID Trimethylamine 1146 C00565 PNNL_Jianzhi_ID Trimethylamine N-oxide 1145 C01104 PNNL_Jianzhi_ID Tryptophan 6305 C00078 PNNL_Jianzhi_ID Tyramine 5610 C00483 PNNL_Jianzhi_ID Tyrosine 6057 C00082 PNNL_Jianzhi_ID UDP-galactose 6857410 PNNL_Jianzhi_ID UDP-glucuronate 17473 C00167 PNNL_Jianzhi_ID Uracil 1174 C00106 PNNL_Jianzhi_ID Uridine 6029 C00299 PNNL_Jianzhi_ID Valine 6287 C00183 PNNL_Jianzhi_ID Xanthine 1188 C00385 PNNL_Jianzhi_ID METABOLITES_END #END