#METABOLOMICS WORKBENCH xujia2018hzau_20180625_223219 DATATRACK_ID:1431 STUDY_ID:ST000982 ANALYSIS_ID:AN001608 PROJECT_ID:PR000671 VERSION 1 CREATED_ON June 26, 2018, 11:08 am #PROJECT PR:PROJECT_TITLE Analysis the concentration of betaine, choline, TMA, TMAO and amino acids in PR:PROJECT_TITLE plasma in Megalobrama amblycephala. PR:PROJECT_SUMMARY Analysis the concentration of betaine, choline, TMA, TMAO and amino acids in PR:PROJECT_SUMMARY plasma in Megalobrama amblycephalab by HPLC-MS/MS PR:INSTITUTE Huazhong Agricultural University PR:LAST_NAME Xu PR:FIRST_NAME Jia PR:ADDRESS Lion rock street, hung shan district, Wuhan, HuBei, 430070, China PR:EMAIL xujia2018hzau@163.com PR:PHONE 13018097215 #STUDY ST:STUDY_TITLE Analysis the concentration of betaine, choline, TMA, TMAO and amino acids in ST:STUDY_TITLE plasma in Megalobrama amblycephala. ST:STUDY_SUMMARY Analysis the concentration of betaine, choline, TMA, TMAO and amino acids in ST:STUDY_SUMMARY plasma in Megalobrama amblycephala by HPLC-MS/MS. ST:INSTITUTE Huazhong Agricultural University ST:LAST_NAME Xu ST:FIRST_NAME Jia ST:ADDRESS Lion rock street, hung shan district, Wuhan, HuBei, 430070, China ST:EMAIL xujia2018hzau@163.com ST:PHONE 13018097215 #SUBJECT SU:SUBJECT_TYPE Fish SU:SUBJECT_SPECIES Megalobrama amblycephala SU:TAXONOMY_ID 75352 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1 Diet:Control group SUBJECT_SAMPLE_FACTORS - 2 Diet:Control group SUBJECT_SAMPLE_FACTORS - 3 Diet:Control group SUBJECT_SAMPLE_FACTORS - 4 Diet:Control group SUBJECT_SAMPLE_FACTORS - 5 Diet:Control group SUBJECT_SAMPLE_FACTORS - 6 Diet:Control group SUBJECT_SAMPLE_FACTORS - 7 Diet:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 8 Diet:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 9 Diet:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 10 Diet:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 11 Diet:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 12 Diet:High carbohydrates group SUBJECT_SAMPLE_FACTORS - 13 Diet:Betaine supplementary for 16 weeks SUBJECT_SAMPLE_FACTORS - 14 Diet:Betaine supplementary for 16 weeks SUBJECT_SAMPLE_FACTORS - 15 Diet:Betaine supplementary for 16 weeks SUBJECT_SAMPLE_FACTORS - 16 Diet:Betaine supplementary for 16 weeks SUBJECT_SAMPLE_FACTORS - 17 Diet:Betaine supplementary for 16 weeks SUBJECT_SAMPLE_FACTORS - 18 Diet:Betaine supplementary for 16 weeks SUBJECT_SAMPLE_FACTORS - 19 Diet:Betaine supplementary for 4 weeks SUBJECT_SAMPLE_FACTORS - 20 Diet:Betaine supplementary for 4 weeks SUBJECT_SAMPLE_FACTORS - 21 Diet:Betaine supplementary for 4 weeks SUBJECT_SAMPLE_FACTORS - 22 Diet:Betaine supplementary for 4 weeks SUBJECT_SAMPLE_FACTORS - 23 Diet:Betaine supplementary for 4 weeks SUBJECT_SAMPLE_FACTORS - 24 Diet:Betaine supplementary for 4 weeks #COLLECTION CO:COLLECTION_SUMMARY After 16 weeks of the feeding trial, approximately 24h after the last feeding, CO:COLLECTION_SUMMARY fish in all tanks were anaesthetized with MS-222 (Sigma Aldrich, USA) at the CO:COLLECTION_SUMMARY concentration of 100 mg L-1. Fifteen specimens from each tank were selected CO:COLLECTION_SUMMARY randomly to obtain the blood. Blood was obtained from the caudal vein using CO:COLLECTION_SUMMARY sterile syringes with pre-added anticoagulant solution and then centrifuged CO:COLLECTION_SUMMARY (3000×g) at 4°C for 10 min to obtain the serum, which was quickly frozen in CO:COLLECTION_SUMMARY liquid nitrogen and stored at -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 betaine TR:TREATMENT_SUMMARY treatment diet (HCB) to contain a high level of carbohydrates (35.64±0.43%) TR:TREATMENT_SUMMARY with 1% betaine supplemented for 16 weeks. HC4B group was fed a combination of TR:TREATMENT_SUMMARY HCD (first twelve weeks) and HCB diets (last four weeks). TR:TREATMENT_PROTOCOL_FILENAME Experiment_methods.docx #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fifty μl of protein precipitant (containing NVL) was added to 50 μl of serum SP:SAMPLEPREP_SUMMARY solution of each sample, mixed thoroughly, and centrifuged at 13200 rpm for 4 SP:SAMPLEPREP_SUMMARY min. The supernatant (10 μl) was mixed with 50 μl methanol borate buffer (0.1 SP:SAMPLEPREP_SUMMARY M, pH=8.8) thoroughly. For amino acids, the above solution was mixed with 20 μl SP:SAMPLEPREP_SUMMARY of diethyl ethoxymethylene-malonate at 55°C for 15 min. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Unspecified #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex API 3200 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS umol/L or ng/mL 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 Diet:Control group Diet:Control group Diet:Control group Diet:Control group Diet:Control group Diet:Control group Diet:High carbohydrates group Diet:High carbohydrates group Diet:High carbohydrates group Diet:High carbohydrates group Diet:High carbohydrates group Diet:High carbohydrates group Diet:Betaine supplementary for 16 weeks Diet:Betaine supplementary for 16 weeks Diet:Betaine supplementary for 16 weeks Diet:Betaine supplementary for 16 weeks Diet:Betaine supplementary for 16 weeks Diet:Betaine supplementary for 16 weeks Diet:Betaine supplementary for 4 weeks Diet:Betaine supplementary for 4 weeks Diet:Betaine supplementary for 4 weeks Diet:Betaine supplementary for 4 weeks Diet:Betaine supplementary for 4 weeks Diet:Betaine supplementary for 4 weeks TMA 496 418 206 544 435 554 112 16.8 676 466 47 110 745 693 39 244 262 271 510 624 326 292 195 198 TMAO 3.96 3.44 4.08 3.71 3.97 3.84 3.21 3.38 3.91 3.14 3.96 2.52 3.32 2.41 2.4 2.18 2.5 2.49 3.26 3.12 2.53 2.59 3.68 2.85 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 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 Ala 780.00 930.00 703.00 743.00 1050.00 610.00 758.00 596.00 742.00 668.00 569.00 774.00 600.00 623.00 469.00 661.00 538.00 526.00 456.00 604.00 691.00 700.00 600.00 503.00 Pro 670.00 935.00 654.00 576.00 824.00 557.00 978.00 830.00 634.00 357.00 844.00 631.00 191.00 194.00 544.00 296.00 446.00 492.00 163.00 261.00 602.00 426.00 780.00 719.00 Val 228.00 383.00 245.00 241.00 209.00 171.00 339.00 229.00 187.00 152.00 266.00 236.00 123.00 130.00 187.00 160.00 157.00 178.00 142.00 157.00 164.00 199.00 190.00 178.00 Ile 95.20 95.40 90.90 102.00 104.00 89.20 172.00 96.20 73.50 64.10 134.00 109.00 50.40 57.70 76.40 59.70 69.20 80.00 54.80 57.70 61.50 70.60 64.10 76.20 Gln 374.00 553.00 360.00 388.00 485.00 325.00 443.00 392.00 370.00 332.00 329.00 283.00 319.00 299.00 323.00 291.00 316.00 327.00 251.00 280.00 299.00 308.00 473.00 367.00 Glu 220.00 264.00 198.00 115.00 281.00 146.00 148.00 62.60 110.00 126.00 32.30 142.00 89.60 116.00 39.90 261.00 36.50 37.00 22.90 97.20 115.00 152.00 102.00 88.60 Phe 128.00 118.00 94.20 80.40 107.00 83.30 113.00 104.00 76.90 70.90 77.30 61.00 100.00 87.00 88.90 79.10 78.00 89.50 67.80 84.10 70.00 74.10 66.40 51.80 Arg 401.00 496.00 308.00 306.00 333.00 231.00 376.00 358.00 308.00 273.00 281.00 297.00 286.00 251.00 276.00 292.00 280.00 290.00 175.00 219.00 293.00 322.00 294.00 294.00 Tyr 167.00 241.00 100.00 95.20 116.00 91.00 128.00 131.00 126.00 120.00 77.30 58.40 151.00 154.00 111.00 121.00 110.00 123.00 96.80 120.00 113.00 134.00 79.80 52.30 Trp 42.10 36.30 32.90 30.50 31.80 25.10 43.80 50.10 35.60 33.90 40.40 25.20 39.60 33.60 37.10 28.30 34.50 42.10 26.60 31.10 26.60 25.30 30.00 22.20 Lys 299.00 392.00 298.00 359.00 518.00 294.00 359.00 261.00 250.00 259.00 270.00 422.00 190.00 195.00 242.00 308.00 243.00 249.00 130.00 228.00 218.00 246.00 208.00 261.00 Leu 166.00 173.00 174.00 194.00 188.00 130.00 237.00 179.00 127.00 102.00 219.00 208.00 80.10 97.60 133.00 113.00 127.00 114.00 93.40 129.00 104.00 131.00 122.00 131.00 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name MRM (precursor ion/fragment ion) Internal Standard Declustering Potential (v) Impact Energy (v) TMA 60.080/44.000 TMAO-d9 (85.050/66.050) 20 15 TMAO 76.080/58.050 TMAO-d9 (85.050/66.050) 20 15 Betaine 118.070/59.100 TMAO-d9 (85.050/66.050) 20 15 Choline 104.300/60.200 TMAO-d9 (85.050/66.050) 20 15 Ala 260.100/171.010 NVL(288.150/171.010) 30 20 Pro 286.150/171.010 NVL(288.150/171.010) 30 20 Val 288.150/171.010 NVL(288.150/171.010) 30 20 Ile 302.150/171.010 NVL(288.150/171.010) 30 20 Gln 317.150/171.010 NVL(288.150/171.010) 30 20 Glu 318.100/171.010 NVL(288.150/171.010) 30 20 Phe 336.200/171.010 NVL(288.150/171.010) 30 20 Arg 345.210/171.010 NVL(288.150/171.010) 30 20 Tyr 352.200/171.010 NVL(288.150/171.010) 30 20 Trp 375.200/171.010 NVL(288.150/171.010) 30 20 Lys 487.200/171.010 NVL(288.150/171.010) 30 20 Leu 302.150/171.010 NVL(288.150/171.010) 30 20 METABOLITES_END #END