#METABOLOMICS WORKBENCH FernandezGarcia_M_20201216_121315 DATATRACK_ID:2361 STUDY_ID:ST001878 ANALYSIS_ID:AN003081 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_1 bdivrou_mero_2_met_1 bdivrou_mero_3_met_1 bdivrou_mero_4_met_1 bdivrou_mero_5_met_1 Factors Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Adenosine-5^-diphosphate 107.5000 200.5000 50.5000 120.5000 20.5000 Adenosine-5^-monophosphate 16314.0000 16645.0000 11816.0000 14199.0000 10432.0000 Arginine 1401.5000 1272.5000 1222.5000 944.5000 1065.5000 Asparagine 13990.5000 15073.5000 12945.5000 14566.5000 13407.5000 Aspartic Acid 214961.5000 208835.5000 136156.5000 177389.5000 115635.5000 Citric acid // Isocitric acid 12593.5000 17132.5000 3339.5000 4934.5000 4485.5000 Citrulline 94667.5000 96647.5000 81869.5000 93635.5000 87903.5000 Dihydroxyacetone phosphate 4221.0000 4886.0000 3173.0000 3237.0000 2061.0000 Fructose-6-phosphate 51506.5000 54039.5000 44090.5000 52555.5000 44455.5000 Fumaric acid 5876.5000 6531.5000 3640.5000 6206.5000 4402.5000 Glucose-6-phosphate 10726.5000 10233.5000 8993.5000 9485.5000 8824.5000 Glutamic acid 579012.0000 567655.0000 400759.0000 511618.0000 344408.0000 Glutamine 21776.0000 22073.0000 19499.0000 21597.0000 21361.0000 Histidine 3826.0000 4168.0000 2908.0000 3789.0000 3126.0000 Isoleucine 989.5000 913.5000 779.5000 542.5000 697.5000 Itaconic acid 1066.0000 2395.0000 1839.0000 371.0000 735.0000 Ketoglutaric acid 32861.0000 29764.0000 21696.0000 27545.0000 16896.0000 Lactic acid 1058508.5000 1105945.5000 848862.5000 987510.5000 872919.5000 Leucine 4478.0000 4227.0000 3706.0000 3866.0000 3232.0000 Malic acid 1000869.0000 939859.0000 637814.0000 775293.0000 477926.0000 Methionine 9446.5000 7841.5000 7415.5000 8830.5000 6691.5000 NAD+ 67911.0000 67172.0000 50383.0000 59179.0000 49838.0000 Phenylalanine 61257.5000 63451.5000 46267.5000 56986.5000 43347.5000 Phosphoenolpyruvic acid 1471.0000 1498.0000 815.0000 902.0000 424.0000 Proline 459.5000 499.5000 349.5000 489.5000 309.5000 Pyruvic acid 985178.0000 1036468.0000 879626.0000 936154.0000 774517.0000 Ribose-5-phosphate 3800.5000 4507.5000 4139.5000 2680.5000 3868.5000 Ribulose-5-phosphate // Xylulose-5-phosphate 13222.0000 14704.0000 10743.0000 10736.0000 8384.0000 Sedoheptulose-7-phosphate 3017.5000 3048.5000 2492.5000 3045.5000 2254.5000 Serine 9001.5000 10473.5000 8442.5000 9213.5000 9430.5000 Succinic acid 209385.0000 249660.0000 141214.0000 203320.0000 145226.0000 Threonine 8782.0000 9106.0000 7658.0000 8834.0000 7935.0000 Tryptophan 40595.5000 44876.5000 38425.5000 43179.5000 36976.5000 Tyrosine 19189.5000 18327.5000 14229.5000 16696.5000 13475.5000 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 Adenosine-5'-diphosphate C00008 Adenosine-5'-monophosphate C00020 Arginine C00062 Asparagine C00152 Aspartic Acid C00049 Citric acid // Isocitric acid C00158 // C00311 Citrulline C00327 Dihydroxyacetone phosphate C00111 Fructose-6-phosphate C00085 Fumaric acid C00122 Glucose-6-phosphate C00668 Glutamic acid C00025 Glutamine C00064 Histidine C00135 Isoleucine C00407 Itaconic acid C00490 Ketoglutaric acid C00026 Lactic acid C00186 Leucine C00123 Malic acid C00149 Methionine C00073 NAD+ C00003 Phenylalanine C00079 Phosphoenolpyruvic acid C00074 Proline C00148 Pyruvic acid C00022 Ribose-5-phosphate C00117 Ribulose-5-phosphate // Xylulose-5-phosphate C00199 // C00231 Sedoheptulose-7-phosphate C05382 Serine C00065 Succinic acid C00042 Threonine C00188 Tryptophan C00078 Tyrosine C00082 METABOLITES_END #END