#METABOLOMICS WORKBENCH FernandezGarcia_M_20201216_121315 DATATRACK_ID:2361 STUDY_ID:ST001878 ANALYSIS_ID:AN003082 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Babesia merozoite targeted metabolomics project PR:PROJECT_TYPE Characterization of the central carbon metabolism and associated pathways PR:PROJECT_SUMMARY The project aims to detect polar metabolites corresponding to metabolites PR:PROJECT_SUMMARY present in isolated merozoites of the apicomplexan parasite Babesia divergens PR:PROJECT_SUMMARY using liquid chromatography coupled to a triple-quad mass spectrometer and PR:PROJECT_SUMMARY defined transitions for target metabolites of relevance PR:INSTITUTE CEU San Pablo University PR:DEPARTMENT Departamento de Quimica y Bioquimica PR:LABORATORY Centro de Metabolomica y Bioanalisis (CEMBIO) PR:LAST_NAME Fernandez PR:FIRST_NAME Miguel PR:ADDRESS Universidad CEU San Pablo, Campus de Montepríncipe, Alcorcón, Madrid, 28925, PR:ADDRESS Spain PR:EMAIL mig.fernandez.ce@ceindo.ceu.es PR:PHONE 913724711 PR:DOI http://dx.doi.org/10.21228/M8NX2V #STUDY ST:STUDY_TITLE Targeted analysis of Babesia divergens merozoites ST:STUDY_SUMMARY The study comprehends two consecutive LC-QqQ/MS analyses of Babesia divergens ST:STUDY_SUMMARY merozoite extracts isolated from B. divergens infected red blood cell cultures ST:STUDY_SUMMARY performed under identical chromatographic conditions and targeting distinct ST:STUDY_SUMMARY transitions corresponding to metabolites from specific pathways including the ST:STUDY_SUMMARY glycolysis, the TCA cycle, the pentose phosphate pathway, purine and pyrimidine ST:STUDY_SUMMARY biosynthesis and amino acid metabolism. ST:INSTITUTE Universidad CEU San Pablo ST:DEPARTMENT Departamento de Quimica y Bioquimica ST:LABORATORY Centro de Metabolomica y Bioanalisis (CEMBIO) ST:LAST_NAME Fernandez ST:FIRST_NAME Miguel ST:ADDRESS Universidad CEU San Pablo ST:EMAIL mig.fernandez.ce@ceindo.ceu.es ST:PHONE 690090778 ST:SUBMIT_DATE 2020-12-16 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Babesia divergens SU:TAXONOMY_ID 32595 SU:GENOTYPE_STRAIN Rouen 1986 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS mero_1 bdivrou_mero_1_met_1 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_1_met_1.d SUBJECT_SAMPLE_FACTORS mero_1 bdivrou_mero_1_met_2 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_1_met_2.d SUBJECT_SAMPLE_FACTORS mero_2 bdivrou_mero_2_met_1 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_2_met_1.d SUBJECT_SAMPLE_FACTORS mero_2 bdivrou_mero_2_met_2 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_2_met_2.d SUBJECT_SAMPLE_FACTORS mero_3 bdivrou_mero_3_met_1 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_3_met_1.d SUBJECT_SAMPLE_FACTORS mero_3 bdivrou_mero_3_met_2 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_3_met_2.d SUBJECT_SAMPLE_FACTORS mero_4 bdivrou_mero_4_met_1 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_4_met_1.d SUBJECT_SAMPLE_FACTORS mero_4 bdivrou_mero_4_met_2 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_4_met_2.d SUBJECT_SAMPLE_FACTORS mero_5 bdivrou_mero_5_met_1 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_5_met_1.d SUBJECT_SAMPLE_FACTORS mero_5 bdivrou_mero_5_met_2 Genotype:Wild-type RAW_FILE_NAME=bdivrou_mero_5_met_2.d #COLLECTION CO:COLLECTION_SUMMARY Merozoites were isolated from B. divergens infected erythrocite (iRBC) cultures CO:COLLECTION_SUMMARY at 40% parasitemia. The content from iRBC culture flasks was transferred to CO:COLLECTION_SUMMARY Falcon tubes and spinned at 600 x g and 4 ºC for 5 min. Then, supernatants were CO:COLLECTION_SUMMARY filtered once using 5 μm filters and twice using 1.2 μm filters (Versapor CO:COLLECTION_SUMMARY membranes) with the help of a 1 mL syringe. Subsequently, the resulting volume CO:COLLECTION_SUMMARY was spinned on a Falcon tube at 2000 x g and 4 ºC for 2 min. The supernatants CO:COLLECTION_SUMMARY were discarded, and merozoite-containing pellets were placed on ice. CO:SAMPLE_TYPE merozoites isolated from RBC #TREATMENT TR:TREATMENT_SUMMARY Since this was a qualitative study aiming to characterize specific metabolites TR:TREATMENT_SUMMARY from the Babesia divergens merozoites, only one sample group was included and no TR:TREATMENT_SUMMARY treatment was performed #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite extraction and quenching: four volumes of cold methanol were added to SP:SAMPLEPREP_SUMMARY one volume isolated B. divergens merozoite pellets placed on ice and quickly SP:SAMPLEPREP_SUMMARY mixed using a high speed vortex. Then, samples were placed in an ice bath for 10 SP:SAMPLEPREP_SUMMARY min, Subsequently, samples were transferred to liquid nitrogen for 10 min and SP:SAMPLEPREP_SUMMARY thawed in an ice bath for 10 min (this freeze-thaw cycle was repeated two SP:SAMPLEPREP_SUMMARY times). Samples were then centrifuged at 5725 x g and 4 ºC for 5 min. SP:SAMPLEPREP_SUMMARY Supernatants containing the metabolite extracts were transferred to new tubes, SP:SAMPLEPREP_SUMMARY while the remaining pellets were re-extracted twice by adding 400 μL of cold SP:SAMPLEPREP_SUMMARY methanol and undergoing the liquid nitrogen freeze-thaw cycles described above. SP:SAMPLEPREP_SUMMARY Supernatants obtained for each biological replicate were combined and filtered SP:SAMPLEPREP_SUMMARY through 0.22 μm nylon syringe filters and stored at -80 ºC until use. Prior to SP:SAMPLEPREP_SUMMARY LC-QqQ/MS analysis, supernatants underwent LC-QqQ/MS-specific sample SP:SAMPLEPREP_SUMMARY preparation. First supernatants were thawed on ice and vortex mixed for 2 min. SP:SAMPLEPREP_SUMMARY On ice, 300 µL of each supernatant were transferred to LC/MS vials and SP:SAMPLEPREP_SUMMARY evaporated using a vacuum concentrator at <10 bar for 2h. The dried extracts SP:SAMPLEPREP_SUMMARY were then reconstituted in 60 µL of Milli-Q water with the help of an SP:SAMPLEPREP_SUMMARY ultrasonic bath for 5 min. Then, samples were subjected to LC-QqQ/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The chromatographic method consists on a subtle adaptation of the Agilent dMRM CH:CHROMATOGRAPHY_SUMMARY method, which uses tributylamine as an ion pairing reagent (for more CH:CHROMATOGRAPHY_SUMMARY information, see CH:CHROMATOGRAPHY_SUMMARY https://www.agilent.com/cs/library/technicaloverviews/public/5991-6482EN.pdf) CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um) CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS Method 1: Data was acquired in MRM mode. To achieve a higher signal from MS:MS_COMMENTS low-abundance metabolites, transitions corresponding to metabolites of interests MS:MS_COMMENTS were separated in two MS methods (see method 2). Transitions utilized in this MS:MS_COMMENTS study are subtle modifications of the transition dataset contained in the MS:MS_COMMENTS Agilent dMRM database and method (see MS:MS_COMMENTS https://www.agilent.com/cs/library/technicaloverviews/public/5991-6482EN.pdf). MS:MS_COMMENTS Feature assignment was performed by matching transitions and RT with these MS:MS_COMMENTS contained in the Agilent dMRM Database and Method, and further confirmed by MS:MS_COMMENTS addition of authentic MS-grade standards. The software workflow included user MS:MS_COMMENTS visualization in Agilent MassHunter Workstation Software Qualitative Analysis MS:MS_COMMENTS (version B.09.00), and further compound integration using Agilent MassHunter MS:MS_COMMENTS Workstation Software Quantitative Analysis (version B.09.00). Only metabolites MS:MS_COMMENTS with signal-to-noise ratio higher than 3 (limit of detection) were reported. MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS blank-substracted abundances MS_METABOLITE_DATA_START Samples bdivrou_mero_1_met_2 bdivrou_mero_2_met_2 bdivrou_mero_3_met_2 bdivrou_mero_4_met_2 bdivrou_mero_5_met_2 Factors Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type 2_3-Dihydroxyisovalerate 2410.0000 2838.0000 2070.0000 2793.0000 1902.0000 2-Deoxycytidine-5^-monophosphate 2149.0000 1911.0000 1462.0000 2053.0000 1337.0000 2-Deoxyuridine 1075.0000 1067.0000 1154.0000 1325.0000 1228.0000 4-Pyridoxic acid 1421.5000 1525.5000 1337.5000 945.5000 1115.5000 5^-Deoxy-5^-(methylthio)adenosine 2856.0000 1421.0000 1680.0000 2985.0000 1841.0000 Adenosine-3^_5^-cyclic monophosphate 561.0000 1569.0000 515.0000 1310.0000 957.0000 Carnitine 525.0000 304.0000 227.0000 328.0000 293.0000 Creatine 3374.0000 3176.0000 2428.0000 2808.0000 2207.0000 Creatinine 675.0000 614.0000 554.0000 820.0000 814.0000 Cytidine-5^-monophosphate 3803.0000 3612.0000 2400.0000 3589.0000 2090.0000 Dihydroorotic acid 1576.0000 1114.0000 634.0000 872.0000 1184.0000 Dihydrosphingosine 2261.5000 1410.5000 1206.5000 2093.5000 1144.5000 gamma-Glutamylcysteine 1641.0000 1725.0000 1840.0000 Gluconic acid // Galactonic acid 769.5000 596.5000 499.5000 556.5000 334.5000 Glucosamine // Galactosamine 528.0000 1127.0000 316.0000 1059.0000 723.0000 Glucose-1-phosphate // Mannose-1-phosphate 2764.0000 1887.0000 1876.0000 2119.0000 1984.0000 Gluthathione (oxidized_ GSSG) 20624.0000 11784.0000 7741.0000 12481.0000 5273.0000 Glyceric acid 4610.0000 3228.0000 2735.0000 2743.0000 1964.0000 Guanine 916.5000 657.5000 423.5000 618.5000 566.5000 Guanosine 2108.0000 1747.0000 1216.0000 1482.0000 1288.0000 Hydroxyhippuric acid 2138.5000 1282.5000 802.5000 1269.5000 964.5000 Hypoxanthine 235797.0000 201272.0000 129019.0000 216173.0000 134377.0000 Indoline carboxylate 338.5000 848.5000 301.5000 110.5000 214.5000 Inosine 33004.0000 26946.0000 19733.0000 29731.0000 21178.0000 Inosine-5^-monophosphate 99144.0000 85968.0000 57616.0000 86931.0000 48697.0000 Inositol 1104653.5000 889014.5000 915508.5000 1100748.5000 949953.5000 Ketoisovaleric acid // Ketovaleric acid 55689.0000 48751.0000 35421.0000 55224.0000 31039.0000 Malonic acid 78502.0000 69729.0000 51996.0000 86915.0000 75221.0000 Mannose // Sorbose 9043.0000 8276.0000 7709.0000 9184.0000 9145.0000 N-Acetyl-glucosamine-6-phosphate // N-Acetyl-glucosamine-1-phosphate 1902.0000 2483.0000 1435.0000 2388.0000 990.0000 N-Acetylglucosamine // N-Acetylgalactosamine 93.0000 59.0000 120.0000 121.0000 176.0000 N-Acetylneuraminic acid 182.0000 298.0000 215.0000 320.0000 242.0000 N-Carbamoyl-DL-aspartic acid 11637.0000 8647.0000 7178.0000 10963.0000 6987.0000 Nicotinic acid 4114.0000 7941.0000 3457.0000 5027.0000 3938.0000 Nicotinic acid mononucleotide 3324.0000 1900.0000 1455.0000 2234.0000 1346.0000 o-glycosyl-glucose (Maltose // Cellobiose) 462.5000 634.5000 332.5000 306.5000 203.5000 Orotic acid 3834.5000 3960.5000 2803.5000 2766.5000 2765.5000 Pantothenic acid 5013.0000 4819.0000 3216.0000 5197.0000 3421.0000 Pentane-1_2_3_4_5-pentol (Arabitol // Xylitol // Ribitol) 139.0000 598.0000 96.0000 230.0000 198.0000 Phenylpyruvic acid 986.0000 949.0000 443.0000 451.0000 428.0000 p-hydroxybenzoic acid 169192.0000 181447.0000 125797.0000 184169.0000 122270.0000 Pyridoxal 5856.0000 6218.0000 3316.0000 5272.0000 3243.0000 Pyridoxal-5-phosphate 4334.0000 1985.0000 1672.0000 2814.0000 1065.0000 S-(5^-Adenosyl)homocysteine 745.0000 579.0000 358.0000 561.0000 399.0000 Taurine 1426.0000 1857.0000 1122.0000 1665.0000 1591.0000 Taurocholic acid 53.5000 56.5000 35.5000 59.5000 42.5000 Thiamine 1412.0000 778.0000 1120.0000 1745.0000 1426.0000 Thymidine 1584.0000 1687.0000 931.0000 1629.0000 1047.0000 Uridine 19358.0000 16206.0000 10107.0000 17895.0000 9972.0000 Uridine-5^-diphosphoglucose // Uridine 5^-diphosphogalactose 2096.0000 1655.0000 997.0000 2265.0000 566.0000 Uridine-5^-monophosphate 20808.0000 17819.0000 13780.0000 19818.0000 11868.0000 Xanthine 13316.0000 10973.0000 5836.0000 10745.0000 5177.0000 Xanthosine 1434.0000 1295.0000 909.0000 757.0000 951.0000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant 2,3-Dihydroxyisovalerate C04039 2-Deoxycytidine-5'-monophosphate C00239 2-Deoxyuridine C00526 4-Pyridoxic acid C00847 5'-Deoxy-5'-(methylthio)adenosine C00170 Adenosine-3',5'-cyclic monophosphate C00575 Carnitine C00318 Creatine C00300 Creatinine C00791 Cytidine-5'-monophosphate C00055 Dihydroorotic acid C00337 Dihydrosphingosine C00836 gamma-Glutamylcysteine C00669 Gluconic acid // Galactonic acid C00257 // C00880 Glucosamine // Galactosamine C00329 // C02262 Glucose-1-phosphate // Mannose-1-phosphate C00103 // C00636 Gluthathione (oxidized, GSSG) C00127 Glyceric acid C00258 Guanine C00242 Guanosine C00387 Hydroxyhippuric acid Hypoxanthine C00262 Indoline carboxylate Inosine C00294 Inosine-5'-monophosphate C00130 Inositol C00137 Ketoisovaleric acid // Ketovaleric acid C01132 // C06255 Malonic acid C00383 Mannose // Sorbose C00159 // C00247 N-Acetyl-glucosamine-6-phosphate // N-Acetyl-glucosamine-1-phosphate C00357 // C04501 N-Acetylglucosamine // N-Acetylgalactosamine C00140 // C01132 N-Acetylneuraminic acid C00270 N-Carbamoyl-DL-aspartic acid C00438 Nicotinic acid C00253 Nicotinic acid mononucleotide C01185 o-glycosyl-glucose (Maltose // Cellobiose) C00185 // C00208 Orotic acid C00295 Pantothenic acid C00864 Pentane-1,2,3,4,5-pentol (Arabitol // Xylitol // Ribitol) C00532 // C00379 // C00474 Phenylpyruvic acid C00166 p-hydroxybenzoic acid C00805 Pyridoxal C00250 Pyridoxal-5-phosphate C00018 S-(5'-Adenosyl)homocysteine C00021 Taurine C00245 Taurocholic acid C05122 Thiamine C00378 Thymidine C00214 Uridine C00299 Uridine-5'-diphosphoglucose // Uridine 5'-diphosphogalactose C00029 // C00052 Uridine-5'-monophosphate C00105 Xanthine C00385 Xanthosine C01762 METABOLITES_END #END