#METABOLOMICS WORKBENCH xujia2018hzau_20180504_223353_mwtab.txt DATATRACK_ID:1399 STUDY_ID:ST000966 ANALYSIS_ID:AN001580 PROJECT_ID:PR000663 VERSION 1 CREATED_ON May 17, 2018, 1:17 pm #PROJECT PR:PROJECT_TITLE Carbohydrates and betaine studies PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Metabolite and gene expression profiles suggest a putative mechanism through PR:PROJECT_SUMMARY which high dietary carbohydrates reduce the content of hepatic betaine in PR:PROJECT_SUMMARY Megalobrama amblycephala PR:INSTITUTE Huazhong Agricultural University PR:LAST_NAME Xu PR:FIRST_NAME Jia PR:ADDRESS 1 Shizishan Road Hongshan District Wuhan PR:EMAIL xujia2018hzau@163.com PR:PHONE 13018097215 #STUDY ST:STUDY_TITLE Metabolite and gene expression profiles suggest a putative mechanism through ST:STUDY_TITLE which high dietary carbohydrates reduce the content of hepatic betaine in ST:STUDY_TITLE Megalobrama amblycephala ST:STUDY_SUMMARY plasma of Megalobrama amblycephala fed with control diet, high carbohydrates ST:STUDY_SUMMARY diet,betaine diet with 16 weeks and betaine diet with 4 weeks. ST:INSTITUTE College of Fisheries Huazhong Agricultural University ST:LAST_NAME Xu ST:FIRST_NAME Jia ST:ADDRESS 1 Shizishan Road Hongshan District Wuhan ST:EMAIL xujia2018hzau@163.com ST:PHONE 13018097215 #SUBJECT SU:SUBJECT_TYPE Fish SU:SUBJECT_SPECIES Megalobrama amblycephala SU:TAXONOMY_ID 75352 SU:GENDER Not applicable #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1 Experimental variables:Control group SUBJECT_SAMPLE_FACTORS - 2 Experimental variables:Control group SUBJECT_SAMPLE_FACTORS - 3 Experimental variables:Control group SUBJECT_SAMPLE_FACTORS - 4 Experimental variables:Control group SUBJECT_SAMPLE_FACTORS - 5 Experimental variables:Control group SUBJECT_SAMPLE_FACTORS - 6 Experimental variables:Control group SUBJECT_SAMPLE_FACTORS - 7 Experimental variables:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 8 Experimental variables:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 9 Experimental variables:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 10 Experimental variables:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 11 Experimental variables:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 12 Experimental variables:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 13 Experimental variables:Betaine group with 16 weeks SUBJECT_SAMPLE_FACTORS - 14 Experimental variables:Betaine group with 16 weeks SUBJECT_SAMPLE_FACTORS - 15 Experimental variables:Betaine group with 16 weeks SUBJECT_SAMPLE_FACTORS - 16 Experimental variables:Betaine group with 16 weeks SUBJECT_SAMPLE_FACTORS - 17 Experimental variables:Betaine group with 16 weeks SUBJECT_SAMPLE_FACTORS - 18 Experimental variables:Betaine group with 16 weeks SUBJECT_SAMPLE_FACTORS - 19 Experimental variables:Betaine group with 4 weeks SUBJECT_SAMPLE_FACTORS - 20 Experimental variables:Betaine group with 4 weeks SUBJECT_SAMPLE_FACTORS - 21 Experimental variables:Betaine group with 4 weeks SUBJECT_SAMPLE_FACTORS - 22 Experimental variables:Betaine group with 4 weeks SUBJECT_SAMPLE_FACTORS - 23 Experimental variables:Betaine group with 4 weeks SUBJECT_SAMPLE_FACTORS - 24 Experimental variables:Betaine group with 4 weeks #COLLECTION CO:COLLECTION_SUMMARY Blood was obtained from the caudal vein using sterile syringes with pre-added CO:COLLECTION_SUMMARY anticoagulant solution and then centrifuged (3000×g) at 4°C for 10 min to CO:COLLECTION_SUMMARY obtain the serum, which was quickly frozen in liquid nitrogen and stored at CO:COLLECTION_SUMMARY -80°C for biochemical assays. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Control diet (CD group) was formulated to contain 27.11±0.38% carbohydrates. TR:TREATMENT_SUMMARY The high-carbohydrate diet (HCD group) was formulated to contain over TR:TREATMENT_SUMMARY 36.75±0.92% of easily digestible carbohydrates (mostly from flour). The TR:TREATMENT_SUMMARY long-term betaine treatment diet (LBD) to contain a high level of carbohydrates TR:TREATMENT_SUMMARY (35.64±0.43%) with 1% betaine supplemented. SBD group (short-term betaine TR:TREATMENT_SUMMARY treatment diet) was fed a combination of HCD (first twelve weeks) and LBD diets TR:TREATMENT_SUMMARY (last four weeks) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Relative metabolites and amino acids were determined using high-performance SP:SAMPLEPREP_SUMMARY liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) on the SP:SAMPLEPREP_SUMMARY Ultimate3000-API 3200Q TRAP (USA). The analyses were performed by the Beijing SP:SAMPLEPREP_SUMMARY Mass Spectrometry Medical Research Co.,Ltd. (Beijing, China). HPLC-MS/MS detail: SP:SAMPLEPREP_SUMMARY Appropriate amount of water was added to serum, mixed thoroughly, and SP:SAMPLEPREP_SUMMARY centrifuged at 13200rpm for 5min. Supernatant was collected, 100μl of it was SP:SAMPLEPREP_SUMMARY mixed with 400μl of methanol thoroughly, centrifuged again, and the resulting SP:SAMPLEPREP_SUMMARY supernatant was subjected to the HPLC-MS/MS. The HPLC-MS/MS system consisted of SP:SAMPLEPREP_SUMMARY a SRD-3600 Solvent Rack with analytical 6-channel vacuum degasser, a DGP-3600A SP:SAMPLEPREP_SUMMARY pump, WPS-3000TSL analytical autosampler, and a tcc-3200 column compartment. SP:SAMPLEPREP_SUMMARY Chromatographic separations were performed on an MSLab C18 column (150×4.6mm, SP:SAMPLEPREP_SUMMARY 5μm). The mobile phase A was 0.1% formic acid in water, and the organic mobile SP:SAMPLEPREP_SUMMARY phase B was 0.1% ammonium formate in acetonitrile with pH=5.8. The solvent was SP:SAMPLEPREP_SUMMARY delivered to the column at a flow rate of 1 ml min−1 as follows: 0-1 minutes SP:SAMPLEPREP_SUMMARY from A-B (90:10) to A-B (90:10); 1-12 minutes from A-B (90:10) to A-B (30:70); SP:SAMPLEPREP_SUMMARY 12-12.1 minutes from A-B (30:70) to A-B (0:100); 12.1-15 minutes from A-B SP:SAMPLEPREP_SUMMARY (0:100) to A-B (0:100); 15-15.1 minutes from A-B (0:100) to A-B (90:10); 15.1-20 SP:SAMPLEPREP_SUMMARY minutes from A-B(90:10) to A-B (90:10). The conditions for mass spectrometry SP:SAMPLEPREP_SUMMARY detection, optimized to obtain the highest signal intensity, were as follows: SP:SAMPLEPREP_SUMMARY mode=positive-ion mode; ion spray voltage=5500V; nebulizer gas pressure=55psi; SP:SAMPLEPREP_SUMMARY curtain gas pressure=20psi; collision gas pressure=medium; turbo gas SP:SAMPLEPREP_SUMMARY temperature=500°C; entrance potential=10V; collision cell exit potential=2V. SP:SAMPLEPREP_SUMMARY Nitrogen gas was used as the collision gas in the multiple reaction monitoring SP:SAMPLEPREP_SUMMARY mode. The data were processed using Analyst software version 1.5.1 (Applied SP:SAMPLEPREP_SUMMARY Biosystems). Metabolites standard solution (Sigma, USA) was subjected to SP:SAMPLEPREP_SUMMARY HPLC-MS/MS using for calibration of the system and quantification of SP:SAMPLEPREP_SUMMARY metabolites. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The HPLC system consisted of a SRD-3600 Solvent Rack with analytical 6-channel CH:CHROMATOGRAPHY_SUMMARY vacuum degasser, a DGP-3600A pump, WPS-3000TSL analytical autosampler, and a CH:CHROMATOGRAPHY_SUMMARY tcc-3200 column compartment. Chromatographic separations were performed on an CH:CHROMATOGRAPHY_SUMMARY MSLab C18 column (150×4.6mm, 5μm). The mobile phase A was 0.1% formic acid in CH:CHROMATOGRAPHY_SUMMARY water, and the organic mobile phase B was 0.1% ammonium formate in acetonitrile CH:CHROMATOGRAPHY_SUMMARY with pH=5.8. The solvent was delivered to the column at a flow rate of 1 ml CH:CHROMATOGRAPHY_SUMMARY min−1 as follows: 0-1 minutes from A-B (90:10) to A-B (90:10); 1-12 minutes CH:CHROMATOGRAPHY_SUMMARY from A-B (90:10) to A-B (30:70); 12-12.1 minutes from A-B (30:70) to A-B CH:CHROMATOGRAPHY_SUMMARY (0:100); 12.1-15 minutes from A-B (0:100) to A-B (0:100); 15-15.1 minutes from CH:CHROMATOGRAPHY_SUMMARY A-B (0:100) to A-B (90:10); 15.1-20 minutes from A-B(90:10) to A-B (90:10). CH:CHROMATOGRAPHY_TYPE Reversed phase MS:INSTRUMENT_NAME - CH:COLUMN_NAME Unspecified #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME new MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/ml or umol/L 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 Factors Experimental variables:Control group Experimental variables:Control group Experimental variables:Control group Experimental variables:Control group Experimental variables:Control group Experimental variables:Control group Experimental variables:High carbohydrates group Experimental variables:High carbohydrates group Experimental variables:High carbohydrates group Experimental variables:High carbohydrates group Experimental variables:High carbohydrates group Experimental variables:High carbohydrates group Experimental variables:Betaine group with 16 weeks Experimental variables:Betaine group with 16 weeks Experimental variables:Betaine group with 16 weeks Experimental variables:Betaine group with 16 weeks Experimental variables:Betaine group with 16 weeks Experimental variables:Betaine group with 16 weeks Experimental variables:Betaine group with 4 weeks Experimental variables:Betaine group with 4 weeks Experimental variables:Betaine group with 4 weeks Experimental variables:Betaine group with 4 weeks Experimental variables:Betaine group with 4 weeks Experimental variables:Betaine group with 4 weeks Betaine 2490 2780 946 2150 2830 1720 1440 1110 2060 2230 536 2470 1420 1400 551 3950 702 821 518 2300 1130 2180 882 1850 Dimethylglycine 245 1720 2180 1750 1570 2150 1860 2100 2360 1900 1600 1610 2200 3180 2080 1930 2200 1780 1510 1620 1380 1580 1400 1650 Sarcosine 48900 19900 28100 16700 16900 9700 8860 13300 18100 16800 17500 10700 16400 17300 12800 14000 17700 14500 10600 15300 16600 12900 14600 10900 S-adenosylmethionine 51.9 108 361 42.9 9.79 88.6 44.6 333 192 186 586 1.32 572 343 881 6.64 861 899 626 505 359 149 207 275 S-adenosylhomocysteine 92.3 146 41.1 110 17.6 144 61.5 85.5 141 81.9 35.2 61.5 83.5 35.2 248 70.3 211 339 281 171 145 145 101 185 Choline 20400 14000 19400 14600 13400 19100 15800 21300 22800 18400 14000 13000 20500 31500 17600 15700 21000 15900 13700 12700 10700 13400 11500 14300 Carnitine 2530 2160 2150 2500 2370 2150 1730 1390 1570 1390 1900 1580 1990 1920 2370 2620 2570 3000 2950 2070 2460 1820 1420 1480 Acylcarnitine 4170 2330 1790 1370 917 1090 1080 1140 1410 1460 2100 973 1710 1690 1230 1120 1530 1200 1090 1210 972 1340 893 936 Glycine 695.00 766.00 590.00 549.00 937.00 624.00 598.00 591.00 737.00 812.00 478.00 563.00 772.00 994.00 460.00 707.00 695.00 718.00 387.00 537.00 537.00 554.00 620.00 525.00 Serine 180.00 262.00 100.00 109.00 145.00 79.50 111.00 134.00 158.00 126.00 93.60 90.80 161.00 121.00 119.00 125.00 194.00 170.00 116.00 138.00 150.00 112.00 132.00 102.00 Homocysteine 2.92 5.46 9.84 6.01 2.99 4.15 3.96 7.10 5.32 5.83 3.67 3.75 9.85 6.29 8.76 3.56 10.80 12.00 9.93 7.09 6.19 4.79 5.55 6.90 Methionine 78.70 95.70 57.20 46.60 52.20 43.50 43.10 49.50 49.40 45.10 36.10 30.70 73.60 49.90 54.10 37.60 59.50 60.90 46.50 43.40 49.00 39.50 42.60 34.30 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Betaine Dimethylglycine Sarcosine S-adenosylmethionine S-adenosylhomocysteine Choline Carnitine Acylcarnitine Glycine Serine Homocysteine Methionine METABOLITES_END #END