#METABOLOMICS WORKBENCH FernandezGarcia_M_20221004_151220 DATATRACK_ID:3490 STUDY_ID:ST003970 ANALYSIS_ID:AN006534 PROJECT_ID:PR002486 VERSION 1 CREATED_ON June 13, 2025, 5:09 am #PROJECT PR:PROJECT_TITLE Evaluation of Babesia divergens metabolism PR:PROJECT_SUMMARY The project aims to evaluate the metabolism of the piroplasmid apicomplexa PR:PROJECT_SUMMARY Babesia divergens through metabolomics and complementary functional assays PR:PROJECT_SUMMARY involving metabolites. PR:INSTITUTE Centro Nacional de Microbiología CNM-ISCIII PR:LAST_NAME Estrella PR:FIRST_NAME Montero PR:ADDRESS Ctra. de Pozuelo, 28, 28222 Majadahonda, Madrid, España PR:EMAIL estrella.montero@isciii.es PR:PHONE +34 918 22 36 57 #STUDY ST:STUDY_TITLE Comprehensive multiplatform mass spectrometry-based metabolomics unveils ST:STUDY_TITLE insights into Babesia divergens metabolism. ST:STUDY_SUMMARY Babesia divergens is a zoonotic parasite belonging to the apicomplexa phyla ST:STUDY_SUMMARY causing human babesiosis. However, its metabolism remains poorly understood as ST:STUDY_SUMMARY scarce biochemical studies have been reported, and no global metabolic network ST:STUDY_SUMMARY reconstructions have been reported up to date. Comprehensive understanding of ST:STUDY_SUMMARY the B. divergens metabolism could reveal novel therapeutic targets. We performed ST:STUDY_SUMMARY a multiplatform analysis combining targeted and non-targeted mass spectrometry ST:STUDY_SUMMARY based metabolomics of samples (pellets, supernatants) obtained from Babesia ST:STUDY_SUMMARY divergens-infected erythrocyte cultures and non-infected culture controls, ST:STUDY_SUMMARY acquired with CE-ESI(+)/MS, GC-CI(+)/MS, LC-ESI(+)/MS, LC-ESI(-)/MS (all ST:STUDY_SUMMARY non-targeted analyses) and LC-ESI(-)/MS (targeted analysis). Profound metabolite ST:STUDY_SUMMARY level changes occur in both the B. divergens-RBC system and its supernatant ST:STUDY_SUMMARY compared to controls, including those related with central carbon metabolism, ST:STUDY_SUMMARY pyrimidine nucleotide biosynthesis, redox metabolism, glycerophospholipid and ST:STUDY_SUMMARY sphingolipid metabolism, amino acid metabolism and nucleotide metabolism. This ST:STUDY_SUMMARY resource serves as a comprehensive dataset describing how the metabolic ST:STUDY_SUMMARY alterations caused by the intraerythrocytic stage of Babesia divergens are ST:STUDY_SUMMARY reflected in different metabolite profiles across multiple ST:STUDY_SUMMARY mass-spectrometry-based platforms. ST:INSTITUTE CEMBIO ST:LAST_NAME Fernández-García ST:FIRST_NAME Miguel ST:ADDRESS Universidad San Pablo-CEU, Urbanización Montepríncipe, 28925, Alcorcón, ST:ADDRESS Madrid, Spain ST:EMAIL mig.fernandez.ce@ceindo.ceu.es ST:PHONE 690090778 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS iRBC1 iRBC1_CE-TOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iRBC1_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC2 iRBC2_CE-TOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iRBC2_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC3 iRBC3_CE-TOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iRBC3_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC4 iRBC4_CE-TOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iRBC4_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC5 iRBC5_CE-TOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iRBC5_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC1 uRBC1_CE-TOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uRBC1_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC2 uRBC2_CE-TOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uRBC2_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC3 uRBC3_CE-TOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uRBC3_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC4 uRBC4_CE-TOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uRBC4_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC5 uRBC5_CE-TOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uRBC5_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS1 iS1_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iS1_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS2 iS2_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iS2_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS3 iS3_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iS3_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS4 iS4_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iS4_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS5 iS5_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=iS5_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uS1 uS1_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uS1_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uS2 uS2_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uS2_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uS3 uS3_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uS3_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uS4 uS4_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uS4_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS uS5 uS5_CE-TOF-MS Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=CE-TOF-MS; RAW_FILE_NAME=uS5_CE-TOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC1 iRBC1_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iRBC1_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC2 iRBC2_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iRBC2_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC3 iRBC3_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iRBC3_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC4 iRBC4_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iRBC4_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC5 iRBC5_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iRBC5_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC1 uRBC1_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=uRBC1_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC2 uRBC2_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=uRBC2_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC3 uRBC3_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=uRBC3_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC4 uRBC4_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=uRBC4_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS uRBC5 uRBC5_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=uRBC5_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS1 iS1_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iS1_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS2 iS2_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iS2_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS3 iS3_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iS3_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS4 iS4_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iS4_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iS5 iS5_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=iS5_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS uS2 uS2_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=uS2_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS uS3 uS3_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=uS3_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS uS4 uS4_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=uS4_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS uS5 uS5_GC-QTOF-MS Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=GC-QTOF-MS; RAW_FILE_NAME=uS5_GC-QTOF-MS.mzML SUBJECT_SAMPLE_FACTORS iRBC1 iRBC1_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iRBC1_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iRBC2 iRBC2_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iRBC2_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iRBC3 iRBC3_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iRBC3_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iRBC4 iRBC4_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iRBC4_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iRBC5 iRBC5_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iRBC5_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uRBC1 uRBC1_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uRBC1_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uRBC2 uRBC2_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uRBC2_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uRBC3 uRBC3_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uRBC3_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uRBC4 uRBC4_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uRBC4_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uRBC5 uRBC5_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uRBC5_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iS1 iS1_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iS1_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iS2 iS2_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iS2_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iS3 iS3_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iS3_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iS4 iS4_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iS4_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iS5 iS5_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=iS5_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uS1 uS1_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uS1_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uS2 uS2_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uS2_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uS3 uS3_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uS3_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uS4 uS4_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uS4_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS uS5 uS5_LC-QTOF-MSpos Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QTOF-MSpos; RAW_FILE_NAME=uS5_LC-QTOF-MSpos.mzML SUBJECT_SAMPLE_FACTORS iRBC1 iRBC1_LC-QTOF-MSneg Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSneg; RAW_FILE_NAME=iRBC1_LC-QTOF-MSneg.mzML SUBJECT_SAMPLE_FACTORS iRBC2 iRBC2_LC-QTOF-MSneg Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSneg; RAW_FILE_NAME=iRBC2_LC-QTOF-MSneg.mzML SUBJECT_SAMPLE_FACTORS iRBC3 iRBC3_LC-QTOF-MSneg Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSneg; RAW_FILE_NAME=iRBC3_LC-QTOF-MSneg.mzML SUBJECT_SAMPLE_FACTORS iRBC4 iRBC4_LC-QTOF-MSneg Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSneg; RAW_FILE_NAME=iRBC4_LC-QTOF-MSneg.mzML SUBJECT_SAMPLE_FACTORS iRBC5 iRBC5_LC-QTOF-MSneg Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QTOF-MSneg; RAW_FILE_NAME=iRBC5_LC-QTOF-MSneg.mzML SUBJECT_SAMPLE_FACTORS uRBC1 uRBC1_LC-QTOF-MSneg Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QTOF-MSneg; RAW_FILE_NAME=uRBC1_LC-QTOF-MSneg.mzML SUBJECT_SAMPLE_FACTORS uRBC3 uRBC3_LC-QTOF-MSneg Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QTOF-MSneg; RAW_FILE_NAME=uRBC3_LC-QTOF-MSneg.mzML SUBJECT_SAMPLE_FACTORS uRBC4 uRBC4_LC-QTOF-MSneg Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QTOF-MSneg; RAW_FILE_NAME=uRBC4_LC-QTOF-MSneg.mzML SUBJECT_SAMPLE_FACTORS uRBC5 uRBC5_LC-QTOF-MSneg Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QTOF-MSneg; RAW_FILE_NAME=uRBC5_LC-QTOF-MSneg.mzML SUBJECT_SAMPLE_FACTORS iRBC3 iRBC3_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=iRBC3_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS iRBC4 iRBC4_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=iRBC4_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS iRBC5 iRBC5_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=iRBC5_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS uRBC2 uRBC2_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=uRBC2_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS uRBC3 uRBC3_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=uRBC3_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS uRBC4 uRBC4_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=uRBC4_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS uRBC5 uRBC5_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=uRBC5_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS iS1 iS1_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=iS1_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS iS2 iS2_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=iS2_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS iS3 iS3_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=iS3_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS iS4 iS4_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=iS4_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS iS5 iS5_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=iS5_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS uS1 uS1_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=uS1_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS uS2 uS2_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=uS2_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS uS3 uS3_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=uS3_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS uS4 uS4_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=uS4_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS uS5 uS5_LC-QqQ-MS A Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS A; RAW_FILE_NAME=uS5_LC-QqQ-MS A.mzML SUBJECT_SAMPLE_FACTORS iRBC1 iRBC1_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=iRBC1_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS iRBC3 iRBC3_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=iRBC3_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS iRBC4 iRBC4_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=iRBC4_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS iRBC5 iRBC5_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:infected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=iRBC5_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS uRBC2 uRBC2_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=uRBC2_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS uRBC3 uRBC3_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=uRBC3_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS uRBC4 uRBC4_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=uRBC4_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS uRBC5 uRBC5_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:uninfected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=uRBC5_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS iS1 iS1_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=iS1_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS iS2 iS2_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=iS2_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS iS3 iS3_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=iS3_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS iS4 iS4_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=iS4_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS iS5 iS5_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from infected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=iS5_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS uS1 uS1_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=uS1_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS uS2 uS2_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=uS2_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS uS3 uS3_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=uS3_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS uS4 uS4_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=uS4_LC-QqQ-MS B.mzML SUBJECT_SAMPLE_FACTORS uS5 uS5_LC-QqQ-MS B Sample source:erythrocyte culture | Sample type:supernatant from uninfected RBC Analytical platform=LC-QqQ-MS B; RAW_FILE_NAME=uS5_LC-QqQ-MS B.mzML #COLLECTION CO:COLLECTION_SUMMARY Human A+ erythrocytes were obtained from healthy volunteer donors at the Blood CO:COLLECTION_SUMMARY Transfusion Center in Madrid, Spain. Donors provided written informed consent CO:COLLECTION_SUMMARY for the use of their blood in research. All procedures were approved by the CO:COLLECTION_SUMMARY center and carried out in accordance with institutional and regulatory CO:COLLECTION_SUMMARY guidelines. Asynchronous cultures of Babesia divergens (Bd Rouen 1986 strain) CO:COLLECTION_SUMMARY were maintained using human A+ erythrocytes at 5% hematocrit in RPMI 1640 medium CO:COLLECTION_SUMMARY (Gibco, Grand Island, NY, USA), supplemented with 10% human serum (The CO:COLLECTION_SUMMARY Interstate Companies, Memphis, TN, USA), 7.5% (w/v) sodium bicarbonate (Lonza CO:COLLECTION_SUMMARY Group Ltd., Basel, Switzerland), and 50 μg/mL hypoxanthine (Sigma-Aldrich, CO:COLLECTION_SUMMARY St. Louis, MO, USA). Control flasks containing uninfected erythrocytes were CO:COLLECTION_SUMMARY incubated under the same conditions using identical medium. All cultures were CO:COLLECTION_SUMMARY maintained at 37 °C in a humidified atmosphere with 5% CO₂. Samples were CO:COLLECTION_SUMMARY collected from independent asynchronous cultures of B. divergens at CO:COLLECTION_SUMMARY approximately 40% parasitemia. Infected cultures were centrifuged at CO:COLLECTION_SUMMARY 600 × g for 5 minutes at 4 °C using fixed-angle rotors to pellet the CO:COLLECTION_SUMMARY cells (iRBC). The pellets, containing a mixture of infected and uninfected CO:COLLECTION_SUMMARY erythrocytes, were washed and centrifuged three times (2,000 × g, 5 CO:COLLECTION_SUMMARY minutes, 4 °C), resuspended in RPMI, and kept on ice. Supernatants (iS) were CO:COLLECTION_SUMMARY obtained from infected cultures by sequential centrifugation at 2,000 × g CO:COLLECTION_SUMMARY and 8,000 × g (5 minutes each, 4 °C), and stored at –80 °C. Control CO:COLLECTION_SUMMARY samples (uRBC and uS) from uninfected cultures were processed in parallel using CO:COLLECTION_SUMMARY the same protocol. Detailed description of sample collection can be found at CO:COLLECTION_SUMMARY DOI: 10.1007/978-1-0716-1681-9_13 CO:SAMPLE_TYPE Red blood cells and culture media #TREATMENT TR:TREATMENT_SUMMARY Since the study was aimed to describe changes ocurring upon an in vitro model of TR:TREATMENT_SUMMARY a natural disease, no treatment was performed. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite extraction was carried out using a multi-step procedure designed to SP:SAMPLEPREP_SUMMARY rapidly quench metabolism, promote cell lysis, and enhance metabolite recovery. SP:SAMPLEPREP_SUMMARY For intracellular extraction, 800 μL of cold (–20 °C) methanol (Thermo SP:SAMPLEPREP_SUMMARY Fisher Scientific, Loughborough, UK) was added to 200 μL of either infected SP:SAMPLEPREP_SUMMARY (iRBC) or uninfected (uRBC) red blood cells. Samples underwent two freeze–thaw SP:SAMPLEPREP_SUMMARY cycles by immersion in liquid nitrogen for 10 minutes, followed by thawing on SP:SAMPLEPREP_SUMMARY ice and light vortexing. After centrifugation at 5,725 × g for 5 minutes at SP:SAMPLEPREP_SUMMARY 4 °C, the supernatants were collected. The residual pellets were re-extracted SP:SAMPLEPREP_SUMMARY twice with 400 μL of cold methanol per step, using the same freeze–thaw and SP:SAMPLEPREP_SUMMARY centrifugation conditions. All supernatants from the same sample were pooled and SP:SAMPLEPREP_SUMMARY stored at –80 °C until analysis. Extracellular samples (iS and uS) were SP:SAMPLEPREP_SUMMARY processed based on the analytical platform. For untargeted CE-TOF/MS, SP:SAMPLEPREP_SUMMARY GC-QTOF/MS, and LC-QTOF/MS analyses, cold acetonitrile (Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific, Loughborough, UK) was used in a 1:4 (v/v) sample-to-solvent ratio. SP:SAMPLEPREP_SUMMARY For targeted LC-QqQ/MS profiling, cold methanol was used in a 1:3 (v/v) ratio. SP:SAMPLEPREP_SUMMARY Following quenching and extraction, supernatants were filtered through SP:SAMPLEPREP_SUMMARY 0.22 μm MS® Nylon syringe filters (Membrane Solutions, Plano, USA) and SP:SAMPLEPREP_SUMMARY stored at –80 °C. For CE-TOF/MS analysis, 250 μL of each extract (iRBC, SP:SAMPLEPREP_SUMMARY uRBC, iS, and uS) was dried under vacuum and reconstituted in 50 μL of CE/MS SP:SAMPLEPREP_SUMMARY sample solution prepared by dissolving methionine sulfone (internal standard; SP:SAMPLEPREP_SUMMARY Sigma-Aldrich, Steinheim, Germany) at 0.2 mM in Milli-Q water with 0.1 M SP:SAMPLEPREP_SUMMARY formic acid (Sigma-Aldrich). After vortexing for 1 minute and centrifugation at SP:SAMPLEPREP_SUMMARY 12,600 × g for 15 minutes at 4 °C, the supernatants were used for analysis. SP:SAMPLEPREP_SUMMARY GC-QTOF/MS samples were prepared by adding 10 μL of ethoxymation solution and SP:SAMPLEPREP_SUMMARY 12 μL of internal standard to 240 μL of each sample. The ethoxymation SP:SAMPLEPREP_SUMMARY reagent was anhydrous pyridine containing 19 mg·mL⁻¹ O-ethoxyamine SP:SAMPLEPREP_SUMMARY (Sigma-Aldrich, Steinheim, Germany), and the internal standard solution SP:SAMPLEPREP_SUMMARY contained 10 mg·L⁻¹ meso-erythritol in Milli-Q water. After vacuum drying, SP:SAMPLEPREP_SUMMARY derivatization was performed automatically using an MPS autosampler. SP:SAMPLEPREP_SUMMARY O-ethyloxime derivatives were formed by incubating the extracts for 90 minutes SP:SAMPLEPREP_SUMMARY at 40 °C, followed by silylation of acidic compounds using 42 μL of SP:SAMPLEPREP_SUMMARY N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% SP:SAMPLEPREP_SUMMARY trimethylchlorosilane (TCMS; Sigma-Aldrich, Germany) for 50 minutes at 40 °C. SP:SAMPLEPREP_SUMMARY For LC-QTOF/MS analysis, 100 μL of each processed extract (iRBC, uRBC, iS, SP:SAMPLEPREP_SUMMARY and uS) was transferred to LC vials after brief vortexing. For LC-QqQ/MS SP:SAMPLEPREP_SUMMARY analysis of polar metabolites, 12 μL of internal standard solution SP:SAMPLEPREP_SUMMARY (50.09 µM p-chlorophenylalanine in Milli-Q water; Sigma-Aldrich, Steinheim, SP:SAMPLEPREP_SUMMARY Germany) were added to 300 μL of each extract — methanol for iRBC/uRBC and SP:SAMPLEPREP_SUMMARY methanol:water (3:1, v/v) for iS/uS. Samples were then evaporated under vacuum, SP:SAMPLEPREP_SUMMARY reconstituted in 60 μL of Milli-Q water with the aid of a 5-minute sonication SP:SAMPLEPREP_SUMMARY bath, and centrifuged at 3,000 × g for 10 minutes at 4 °C before injection. SP:SAMPLEPREP_SUMMARY Detailed protocols of the conditions followed sample preparation can be found at SP:SAMPLEPREP_SUMMARY DOI: 10.1007/978-1-0716-1681-9_13 #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY non-targeted, LC-MS method for the determination of compounds with intermediate CH:CHROMATOGRAPHY_SUMMARY polarity CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1200 CH:COLUMN_NAME Supelco Discovery HS C18 (150 × 2.1 mm, 3 um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 25 %B at 0 min, increased until 95% B at 35 min. Return to 25 %B at 36 min, CH:FLOW_GRADIENT which was held until 45 min CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 40 ºC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MS method associated with LC-ESI(-)-QTOF/MS Capillary voltage = 3.5 kV Drying MS:MS_COMMENTS gas flow rate = 10.5 L/min Drying gas temperature = 52 psi Fragmentor, skimmer, MS:MS_COMMENTS octopole = 175, 65, 750 V acquisition range = 50-1100 m/z acquisition rate = 1.2 MS:MS_COMMENTS spectra/s reference masses for online m/z correction = 112.9856, 966.0007, MS:MS_COMMENTS 119.0363 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS normalized (total useful signal) abundances MS_METABOLITE_DATA_START Samples iRBC1_LC-QTOF-MSneg iRBC2_LC-QTOF-MSneg iRBC3_LC-QTOF-MSneg iRBC4_LC-QTOF-MSneg iRBC5_LC-QTOF-MSneg uRBC1_LC-QTOF-MSneg uRBC3_LC-QTOF-MSneg uRBC4_LC-QTOF-MSneg uRBC5_LC-QTOF-MSneg Factors Sample source:erythrocyte culture | Sample type:infected RBC Sample source:erythrocyte culture | Sample type:infected RBC Sample source:erythrocyte culture | Sample type:infected RBC Sample source:erythrocyte culture | Sample type:infected RBC Sample source:erythrocyte culture | Sample type:infected RBC Sample source:erythrocyte culture | Sample type:uninfected RBC Sample source:erythrocyte culture | Sample type:uninfected RBC Sample source:erythrocyte culture | Sample type:uninfected RBC Sample source:erythrocyte culture | Sample type:uninfected RBC 3b,16a-Dihydroxyandrostenone sulfate NaN NaN NaN NaN NaN 0.0006 0.0006 0.0006 0.0006 PC(14:0_0:0) 0.0061 0.0065 0.0069 0.0068 0.0079 0.0015 0.0014 0.0015 0.0016 PC(16:1/0:0) 0.0208 0.0305 0.0296 0.0219 0.0156 0.0055 0.0058 0.0077 0.0048 PC(0:0/16:1) 0.0032 0.0019 0.0022 0.0025 0.0028 0.0023 0.0009 0.0006 0.0009 PC(0:0/17:0) 0.0089 0.0084 0.0082 0.0077 0.0078 0.0032 0.0029 0.0033 0.0033 PC(0:0/18:1) 0.0192 0.0292 0.0283 0.0205 0.0132 0.0042 0.0046 0.0066 0.0033 PC(19:0_0:0) 0.0038 0.0049 0.0049 0.0040 0.0031 0.0008 0.0008 0.0010 0.0006 PC(20:0_0:0) 0.0031 0.0036 0.0034 0.0029 0.0020 0.0287 0.0291 0.0319 0.0261 PC(20:3_0:0) 0.0146 0.0119 0.0094 0.0261 0.0213 0.0058 0.0080 0.0056 0.0065 PC(O-18:0_0:0) 0.0269 0.0300 0.0320 0.0277 0.0348 0.0076 0.0072 0.0071 0.0078 PE(18:0/0:0) 0.0019 0.0016 0.0017 0.0016 0.0016 0.0000 0.0000 0.0000 0.0000 PE(22:4_0:0) 0.0029 0.0039 0.0029 0.0028 0.0028 0.0006 0.0005 0.0007 0.0007 PE(22:5_0:0) 0.0145 0.0161 0.0160 0.0155 0.0141 0.0022 0.0017 0.0017 0.0014 PI(18:0_0:0) 0.0010 0.0016 0.0015 0.0010 0.0003 0.0003 0.0003 0.0005 0.0003 PI(20:2_0:0) NaN NaN NaN NaN NaN 0.0005 0.0005 0.0006 0.0004 PC(16:0/8:1);O3 0.0010 NaN NaN 0.0004 0.0017 NaN 0.0026 NaN NaN MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG COMPOUND ID Molecular feature or transition calculated M RT ppm error 3b,16a-Dihydroxyandrostenone sulfate NA 368.166@6.15 368.166 6.15 0.54 PC(14:0_0:0) C04230 // C04233 513.307@15.31 513.307 15.31 0.59 PC(16:1/0:0) C04233 539.322@16.41 539.322 16.41 -0.56 PC(0:0/16:1) C04230 525.342@20.48 525.342 20.48 -2.1 PC(0:0/17:0) C04230 555.347@20.92 555.347 20.92 -11.91 PC(0:0/18:1) C04230 // C04233 567.353@20.3 567.353 20.3 -1.06 PC(19:0_0:0) C04230 // C04233 583.385@25.94 583.385 25.94 0.17 PC(20:0_0:0) C04230 // C04233 597.399@28.19 597.399 28.19 -2.68 PC(20:3_0:0) C04230 // C04233 591.352@19.23 591.352 19.23 -2.71 PC(O-18:0_0:0) 555.389@24.87 555.389 24.87 -1.8 PE(18:0/0:0) C05973 481.317@22.57 481.317 22.57 0.21 PE(22:4_0:0) C04438 // C05973 529.318@20.57 529.318 20.57 2.08 PE(22:5_0:0) C04438 // C05973 527.301@18.54 527.301 18.54 -0.38 PI(18:0_0:0) NA 600.328@25.97 600.328 25.97 0.83 PI(20:2_0:0) NA 670.333@22.73 670.333 22.73 0 PC(16:0/8:1);O3 NA 711.396@26.69 711.396 26.69 0.14 METABOLITES_END #END