#METABOLOMICS WORKBENCH stephensondj_20240227_094013 DATATRACK_ID:4676 STUDY_ID:ST003108 ANALYSIS_ID:AN005088 PROJECT_ID:PR001931 VERSION 1 CREATED_ON February 27, 2024, 10:57 am #PROJECT PR:PROJECT_TITLE Complete absence of GLUT1 does not impair human terminal erythroid PR:PROJECT_TITLE differentiation PR:PROJECT_SUMMARY The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within PR:PROJECT_SUMMARY the erythrocyte membrane and is required for glucose and dehydroascorbic acid PR:PROJECT_SUMMARY (Vitamin C precursor) transport. It is widely recognized as a key protein for PR:PROJECT_SUMMARY red cell structure, function, and metabolism. Previous reports highlighted the PR:PROJECT_SUMMARY importance of GLUT1 activity within these uniquely glycolysis-dependent cells, PR:PROJECT_SUMMARY in particular for increasing antioxidant capacity needed to avoid irreversible PR:PROJECT_SUMMARY damage from oxidative stress in humans. However, studies of glucose transporter PR:PROJECT_SUMMARY roles in erythroid cells are complicated by species-specific differences between PR:PROJECT_SUMMARY humans and mice. Here, using CRISPRmediated gene editing of immortalized PR:PROJECT_SUMMARY erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate PR:PROJECT_SUMMARY committed human erythroid cells completely deficient in expression of GLUT1. We PR:PROJECT_SUMMARY show that absence of GLUT1 does not impede human erythroblast proliferation, PR:PROJECT_SUMMARY differentiation, or enucleation. This work demonstrates for the first-time PR:PROJECT_SUMMARY generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient PR:PROJECT_SUMMARY reticulocytes possess no tangible alterations to membrane composition or PR:PROJECT_SUMMARY deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient PR:PROJECT_SUMMARY reticulocytes reveal hallmarks of reduced glucose import, downregulated PR:PROJECT_SUMMARY metabolic processes and upregulated AMPK-signalling, alongside alterations in PR:PROJECT_SUMMARY antioxidant metabolism, resulting in increased osmotic fragility and metabolic PR:PROJECT_SUMMARY shifts indicative of higher oxidant stress. Despite detectable metabolic changes PR:PROJECT_SUMMARY in GLUT1 deficient reticulocytes, the absence of developmental phenotype, PR:PROJECT_SUMMARY detectable proteomic compensation or impaired deformability comprehensively PR:PROJECT_SUMMARY alters our understanding of the role of GLUT1 in red blood cell structure, PR:PROJECT_SUMMARY function and metabolism. It also provides cell biological evidence supporting PR:PROJECT_SUMMARY clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 PR:PROJECT_SUMMARY deficiency syndrome. PR:INSTITUTE University of Colorado PR:LAST_NAME Stephenson PR:FIRST_NAME Daniel PR:ADDRESS Research 1 South L18-1303 12801 E. 17th Ave., Aurora, Colorado, 80045, USA PR:EMAIL daniel.stephenson@cuanschutz.edu PR:PHONE 303-724-3339 #STUDY ST:STUDY_TITLE Complete absence of GLUT1 does not impair human terminal erythroid ST:STUDY_TITLE differentiation ST:STUDY_SUMMARY The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within ST:STUDY_SUMMARY the erythrocyte membrane and is required for glucose and dehydroascorbic acid ST:STUDY_SUMMARY (Vitamin C precursor) transport. It is widely recognized as a key protein for ST:STUDY_SUMMARY red cell structure, function, and metabolism. Previous reports highlighted the ST:STUDY_SUMMARY importance of GLUT1 activity within these uniquely glycolysis-dependent cells, ST:STUDY_SUMMARY in particular for increasing antioxidant capacity needed to avoid irreversible ST:STUDY_SUMMARY damage from oxidative stress in humans. However, studies of glucose transporter ST:STUDY_SUMMARY roles in erythroid cells are complicated by species-specific differences between ST:STUDY_SUMMARY humans and mice. Here, using CRISPRmediated gene editing of immortalized ST:STUDY_SUMMARY erythroblasts and adult CD34+ hematopoietic progenitor cells, we generate ST:STUDY_SUMMARY committed human erythroid cells completely deficient in expression of GLUT1. We ST:STUDY_SUMMARY show that absence of GLUT1 does not impede human erythroblast proliferation, ST:STUDY_SUMMARY differentiation, or enucleation. This work demonstrates for the first-time ST:STUDY_SUMMARY generation of enucleated human reticulocytes lacking GLUT1. The GLUT1-deficient ST:STUDY_SUMMARY reticulocytes possess no tangible alterations to membrane composition or ST:STUDY_SUMMARY deformability in reticulocytes. Metabolomic analyses of GLUT1-deficient ST:STUDY_SUMMARY reticulocytes reveal hallmarks of reduced glucose import, downregulated ST:STUDY_SUMMARY metabolic processes and upregulated AMPK-signalling, alongside alterations in ST:STUDY_SUMMARY antioxidant metabolism, resulting in increased osmotic fragility and metabolic ST:STUDY_SUMMARY shifts indicative of higher oxidant stress. Despite detectable metabolic changes ST:STUDY_SUMMARY in GLUT1 deficient reticulocytes, the absence of developmental phenotype, ST:STUDY_SUMMARY detectable proteomic compensation or impaired deformability comprehensively ST:STUDY_SUMMARY alters our understanding of the role of GLUT1 in red blood cell structure, ST:STUDY_SUMMARY function and metabolism. It also provides cell biological evidence supporting ST:STUDY_SUMMARY clinical consensus that reduced GLUT1 expression does not cause anaemia in GLUT1 ST:STUDY_SUMMARY deficiency syndrome. ST:INSTITUTE University of Colorado ST:LAST_NAME Stephenson ST:FIRST_NAME Daniel ST:ADDRESS Research 1 South L18-1303 12801 E. 17th Ave., Aurora, Colorado, 80045, USA ST:EMAIL daniel.stephenson@cuanschutz.edu ST:PHONE 303-724-3339 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 Donor 1 DS1-095-001+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-001+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 1 DS1-095-002+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-002+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 4 DS1-095-003+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-003+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 4 DS1-095-004+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-004+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 5 DS1-095-005+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-005+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 5 DS1-095-006+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-006+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor A DS1-095-007+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-007+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor C DS1-095-008+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-008+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor D DS1-095-009+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-009+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor A DS1-095-010+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-010+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor C DS1-095-011+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-011+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor D DS1-095-012+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-012+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor A DS1-095-013+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-013+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor C DS1-095-014+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-014+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor D DS1-095-015+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-015+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor A DS1-095-016+ Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-016+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor C DS1-095-017+ Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-017+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor D DS1-095-018+ Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-018+.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 1 DS1-095-001- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-001-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 1 DS1-095-002- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-002-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 4 DS1-095-003- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-003-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 4 DS1-095-004- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-004-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 5 DS1-095-005- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-005-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 5 DS1-095-006- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-006-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor A DS1-095-007- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-007-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor C DS1-095-008- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-008-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor D DS1-095-009- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-009-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor A DS1-095-010- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-010-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor C DS1-095-011- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-011-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor D DS1-095-012- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-012-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor A DS1-095-013- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-013-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor C DS1-095-014- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-014-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor D DS1-095-015- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-015-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor A DS1-095-016- Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-016-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor C DS1-095-017- Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-017-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor D DS1-095-018- Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-018-.mzXML; Pellet size=10 Million; Analysis=Metabolomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 1 DS1-095-001L+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-001L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 1 DS1-095-002L+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-002L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 4 DS1-095-003L+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-003L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 4 DS1-095-004L+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-004L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 5 DS1-095-005L+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-005L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 5 DS1-095-006L+ Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-006L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor A DS1-095-007L+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-007L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor C DS1-095-008L+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-008L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor D DS1-095-009L+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-009L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor A DS1-095-010L+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-010L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor C DS1-095-011L+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-011L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor D DS1-095-012L+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-012L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor A DS1-095-013L+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-013L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor C DS1-095-014L+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-014L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor D DS1-095-015L+ Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-015L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor A DS1-095-016L+ Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-016L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor C DS1-095-017L+ Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-017L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor D DS1-095-018L+ Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-018L+.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Positive SUBJECT_SAMPLE_FACTORS Donor 1 DS1-095-001L- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-001L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 1 DS1-095-002L- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-002L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 4 DS1-095-003L- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-003L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 4 DS1-095-004L- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-004L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 5 DS1-095-005L- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-005L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor 5 DS1-095-006L- Sample source:cultured reticulocytes | Assay:04CF29 RAW_FILE_NAME(Raw File Name)=DS1-095-006L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor A DS1-095-007L- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-007L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor C DS1-095-008L- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-008L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor D DS1-095-009L- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-009L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor A DS1-095-010L- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-010L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor C DS1-095-011L- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-011L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor D DS1-095-012L- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-012L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor A DS1-095-013L- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-013L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor C DS1-095-014L- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-014L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor D DS1-095-015L- Sample source:cultured reticulocytes | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-015L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor A DS1-095-016L- Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-016L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor C DS1-095-017L- Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-017L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative SUBJECT_SAMPLE_FACTORS Donor D DS1-095-018L- Sample source:Red blood cells | Assay:01CF19 RAW_FILE_NAME(Raw File Name)=DS1-095-018L-.RAW; Pellet size=10 Million; Analysis=Lipidomics; Polarity=Negative #COLLECTION CO:COLLECTION_SUMMARY For flow cytometry, cells were fixed (1% paraformaldehyde, 0.0075% CO:COLLECTION_SUMMARY glutaraldehyde) to prevent antibody agglutination, except for assays using CO:COLLECTION_SUMMARY GLUT1.RBD. Samples were labelled with primary antibodies in PBSAG (PBS with 1% CO:COLLECTION_SUMMARY (w/v) Glucose and 0.5%(w/v) |Bovine Serum Albumin (BSA)) supplemented with extra CO:COLLECTION_SUMMARY 1%(w/v) BSA for 30min, in the dark. METAFORA’s GLUT1.RBD was incubated at CO:COLLECTION_SUMMARY 37°C and all remaining antibodies incubated at 4°C. Cells were washed 2x PBSAG CO:COLLECTION_SUMMARY and, if required, incubated with appropriate secondary antibody under the same CO:COLLECTION_SUMMARY conditions as described for primary. Cells were washed 2x with PBSAG and CO:COLLECTION_SUMMARY analysed on a Miltenyi MACSQuant 10 flow cytometer. Data was analysed using CO:COLLECTION_SUMMARY FlowJo v10.7 (FlowJo). Reticulocytes were identified by gating on the CO:COLLECTION_SUMMARY Hoechst-negative population. 4 Cells were sorted using a BD Influx Cell Sorter CO:COLLECTION_SUMMARY (BD Biosciences). BEL-A CRISPR edited populations were single cell sorted based CO:COLLECTION_SUMMARY on viability (DRAQ7 negativity). Primary GLUT1 KO cultures were sorted on days 6 CO:COLLECTION_SUMMARY or 7 of differentiation using DRAQ7 and the eGFP-fused GLUT1.RBD to purify the CO:COLLECTION_SUMMARY negative population with a gate based on non-targeting guide control cells. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Filtered reticulocytes (1-2 x105/well) were incubated in decreasing NaCl TR:TREATMENT_SUMMARY concentrations (0.9–0%) for 10min at 37°C. Lysis was stopped by adding 4x TR:TREATMENT_SUMMARY volume of PBSAG. Live cells, considered as having a normal FSC/SSC profile as TR:TREATMENT_SUMMARY defined by the 0.9% NaCl control, were counted by flow cytometry using the TR:TREATMENT_SUMMARY MACSQuant10 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Extraction of metabolites and lipids from red cell pellets was as follows: 36 SP:SAMPLEPREP_SUMMARY µL of cold MeOH:MeCN:H2O (5:3:2, v:v:v) was added to all pellets (pellet size SP:SAMPLEPREP_SUMMARY pre-determined to be 10 million prior to extraction). Samples were vortexed at SP:SAMPLEPREP_SUMMARY 4°C for 30 minutes. Insoluble material was pelleted by centrifugation (18000g, SP:SAMPLEPREP_SUMMARY 10 min) and supernatants were isolated for analysis by UHPLC-MS for metabolomics SP:SAMPLEPREP_SUMMARY and lipidomics. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Metabolomics Negative CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) CH:SOLVENT_A 5% ACN 95% Water 1mM Ammonium Acetate CH:SOLVENT_B 95% ACN 5% Water 1mM Ammonium Acetate CH:FLOW_GRADIENT 0 min - 0.45 ml/min - 0% B, 0.5 min - 0.45ml/min - 0% B, 1.1 min - 0.45ml/min - CH:FLOW_GRADIENT 100% B, 2.75 min - 0.45ml/min - 100% B, 3 min - 0.45ml/min - 0% B, 5min - CH:FLOW_GRADIENT 0.45ml/min - 0%B CH:FLOW_RATE 0.45 ml/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS El-Maven used for data analysis #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS area MS_METABOLITE_DATA_START Samples DS1-095-001- DS1-095-002- DS1-095-003- DS1-095-004- DS1-095-005- DS1-095-006- DS1-095-007- DS1-095-008- DS1-095-009- DS1-095-010- DS1-095-011- DS1-095-012- DS1-095-013- DS1-095-014- DS1-095-015- DS1-095-016- DS1-095-017- DS1-095-018- Factors Sample source:cultured reticulocytes | Assay:04CF29 Sample source:cultured reticulocytes | Assay:04CF29 Sample source:cultured reticulocytes | Assay:04CF29 Sample source:cultured reticulocytes | Assay:04CF29 Sample source:cultured reticulocytes | Assay:04CF29 Sample source:cultured reticulocytes | Assay:04CF29 Sample source:cultured reticulocytes | Assay:01CF19 Sample source:cultured reticulocytes | Assay:01CF19 Sample source:cultured reticulocytes | Assay:01CF19 Sample source:cultured reticulocytes | Assay:01CF19 Sample source:cultured reticulocytes | Assay:01CF19 Sample source:cultured reticulocytes | Assay:01CF19 Sample source:cultured reticulocytes | Assay:01CF19 Sample source:cultured reticulocytes | Assay:01CF19 Sample source:cultured reticulocytes | Assay:01CF19 Sample source:Red blood cells | Assay:01CF19 Sample source:Red blood cells | Assay:01CF19 Sample source:Red blood cells | Assay:01CF19 L-aspartate 2.93E+06 2.04E+06 1.64E+06 3.36E+06 2.37E+06 1.57E+06 2.62E+06 2.50E+06 1.88E+06 7.54E+06 6.48E+06 2.63E+06 5.99E+06 6.09E+06 3.92E+06 2.61E+06 2.64E+06 2.50E+06 L-glutamate 4.17E+06 2.22E+06 2.86E+06 3.46E+06 4.07E+06 1.71E+06 3.47E+06 2.83E+06 1.89E+06 8.00E+06 4.27E+06 3.29E+06 5.42E+06 5.53E+06 4.76E+06 5.96E+05 3.37E+05 1.33E+06 ATP 1.28E+06 1.29E+06 1.16E+06 1.22E+06 1.25E+06 1.52E+06 1.06E+06 1.40E+06 1.40E+06 1.55E+06 1.72E+06 2.03E+06 1.77E+06 2.50E+06 1.48E+06 9.66E+05 1.36E+06 4.92E+05 Cytidine 2.28E+04 1.57E+04 5.24E+03 1.11E+04 2.67E+04 1.06E+04 7.86E+03 1.52E+04 9.30E+03 5.51E+04 2.37E+04 6.38E+03 1.79E+04 2.97E+04 1.67E+04 0.00E+00 0.00E+00 4.58E+03 UMP 1.05E+04 2.32E+04 5.38E+03 2.02E+04 2.56E+03 1.37E+04 1.59E+04 2.07E+04 1.56E+04 6.38E+04 4.86E+04 2.54E+04 9.41E+04 7.17E+04 5.90E+04 5.61E+02 4.06E+02 0.00E+00 Uracil 2.77E+05 1.12E+05 1.15E+05 1.01E+05 1.24E+05 7.25E+04 1.83E+05 1.86E+05 5.43E+04 5.54E+05 5.58E+05 3.88E+04 1.24E+05 1.07E+05 7.81E+04 2.37E+04 2.29E+04 3.78E+04 ITP 6.08E+02 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 1.55E+03 4.28E+03 0.00E+00 0.00E+00 0.00E+00 0.00E+00 8.28E+03 7.34E+03 0.00E+00 0.00E+00 0.00E+00 2.30E+03 IMP 3.84E+05 6.64E+04 1.25E+05 4.22E+04 8.66E+04 2.27E+04 1.33E+05 1.06E+05 3.83E+04 3.94E+05 3.22E+05 3.93E+04 1.34E+05 5.94E+04 9.97E+04 1.19E+05 6.94E+04 1.43E+05 Inosine 2.59E+05 1.07E+05 7.00E+04 6.19E+04 5.60E+04 1.96E+04 2.47E+04 2.65E+04 1.42E+04 5.39E+04 6.28E+04 2.44E+04 4.91E+04 7.03E+04 4.81E+04 9.96E+04 1.15E+05 1.40E+05 4-Pyridoxate 3.24E+04 2.55E+04 1.51E+04 3.00E+04 3.44E+04 3.45E+04 2.22E+04 4.58E+03 1.90E+04 4.41E+04 2.94E+04 5.53E+04 3.53E+04 2.41E+04 4.82E+04 1.33E+04 7.43E+03 2.26E+04 UDP-glucose 2.52E+05 2.40E+05 2.05E+05 2.74E+05 3.00E+05 3.28E+05 1.96E+05 2.01E+05 2.21E+05 4.82E+05 3.98E+05 4.07E+05 5.74E+05 4.69E+05 4.64E+05 1.48E+05 8.85E+04 1.50E+05 Phosphate 3.27E+07 2.64E+07 2.66E+07 4.05E+07 2.92E+07 2.81E+07 2.66E+07 2.45E+07 3.20E+07 2.91E+07 2.45E+07 3.97E+07 2.97E+07 4.80E+07 2.32E+07 4.17E+07 4.53E+07 3.62E+07 Diphosphate 2.12E+06 2.03E+06 1.93E+06 2.88E+06 1.79E+06 2.48E+06 1.78E+06 1.20E+06 2.55E+06 7.75E+05 8.52E+05 3.15E+06 1.68E+06 2.75E+06 1.09E+06 4.27E+06 4.40E+06 3.22E+06 D-Glucose 4.33E+06 2.11E+06 2.41E+06 2.54E+06 4.00E+06 1.90E+06 5.08E+06 6.10E+06 6.46E+06 6.47E+06 4.16E+06 8.13E+06 5.33E+06 8.34E+06 3.53E+06 7.13E+06 4.86E+06 7.59E+06 D-Hexose-phosphate 1.08E+06 1.18E+06 7.31E+05 1.72E+06 1.42E+06 1.36E+06 1.88E+06 9.20E+05 1.33E+06 9.50E+05 8.65E+05 1.50E+06 1.36E+06 1.34E+06 1.25E+06 9.55E+05 1.22E+06 4.64E+05 Phosphoenolpyruvate 5.70E+05 9.76E+05 3.52E+05 1.12E+06 4.79E+05 8.29E+05 8.07E+05 8.71E+05 7.96E+05 7.95E+05 7.23E+05 9.79E+05 1.37E+06 2.24E+06 7.79E+05 5.69E+04 7.47E+04 3.89E+04 Pyruvate 2.80E+05 2.19E+05 2.58E+05 2.83E+05 3.43E+05 2.23E+05 5.61E+05 3.47E+05 5.31E+05 3.44E+05 1.94E+05 3.17E+05 2.57E+05 2.27E+05 1.02E+05 5.37E+05 2.33E+05 6.58E+05 Lactate 2.77E+06 3.60E+06 1.43E+06 3.55E+06 2.32E+06 2.91E+06 6.03E+06 4.24E+06 5.33E+06 5.48E+06 3.35E+06 6.77E+06 6.18E+06 7.31E+06 3.86E+06 4.55E+06 6.14E+06 3.09E+06 Ribose/Ribulose/Arabinose/Xylose/Xylulose 4.75E+05 3.48E+05 2.80E+05 3.62E+05 4.48E+05 2.55E+05 6.30E+05 4.73E+05 2.70E+05 1.25E+06 8.23E+05 3.53E+05 6.42E+05 5.29E+05 4.16E+05 3.97E+05 3.32E+05 4.19E+05 Citrate 9.72E+06 1.04E+07 1.44E+07 1.11E+07 1.68E+07 1.08E+07 3.10E+07 1.75E+07 2.57E+07 1.06E+07 5.32E+06 5.05E+06 1.17E+07 6.62E+06 2.54E+06 1.75E+05 6.19E+05 1.01E+06 Succinate 1.60E+05 1.52E+05 1.35E+05 1.69E+05 1.71E+05 1.16E+05 2.02E+05 1.27E+05 1.65E+05 1.18E+05 1.17E+05 1.92E+05 1.13E+05 1.40E+05 7.79E+04 1.73E+05 1.71E+05 1.84E+05 Fumarate 3.34E+05 3.20E+05 2.30E+05 4.80E+05 3.04E+05 3.04E+05 3.46E+05 2.83E+05 3.04E+05 4.62E+05 3.28E+05 4.37E+05 5.61E+05 5.31E+05 3.25E+05 1.11E+05 1.68E+05 1.54E+05 Malate 2.08E+06 1.64E+06 1.23E+06 2.69E+06 1.53E+06 1.64E+06 1.66E+06 1.32E+06 1.53E+06 1.83E+06 1.39E+06 2.14E+06 2.32E+06 2.83E+06 1.49E+06 4.56E+05 6.97E+05 5.65E+05 2-Hydroxyglutarate 1.67E+06 9.27E+05 8.47E+05 1.03E+06 1.35E+06 1.05E+06 9.68E+05 7.77E+05 8.84E+05 7.00E+05 6.39E+05 9.61E+05 1.44E+06 1.65E+06 7.39E+05 2.75E+05 4.54E+05 5.75E+05 6-Phospho-D-gluconate 1.45E+05 1.51E+05 1.29E+05 2.10E+05 1.99E+05 1.92E+05 2.24E+05 1.68E+05 1.91E+05 1.76E+05 1.72E+05 2.45E+05 1.82E+05 3.15E+05 1.67E+05 1.04E+05 1.98E+05 9.75E+04 Glutathione 7.05E+06 2.16E+06 4.18E+06 2.30E+06 4.97E+06 1.67E+06 3.54E+06 4.35E+06 2.84E+06 1.49E+07 1.02E+07 2.77E+06 5.77E+06 3.62E+06 6.91E+06 2.45E+06 1.43E+06 4.44E+06 Glutathione disulfide 2.92E+05 2.23E+05 2.99E+05 5.22E+05 3.79E+05 4.29E+05 3.74E+05 2.54E+05 4.00E+05 3.63E+05 1.58E+05 2.72E+05 5.56E+05 2.25E+05 4.86E+05 1.40E+06 4.73E+05 1.80E+06 5-Oxoproline 1.75E+06 5.82E+05 8.59E+05 6.84E+05 1.12E+06 3.12E+05 1.15E+06 1.03E+06 4.10E+05 5.90E+06 3.74E+06 6.93E+05 1.87E+06 1.07E+06 1.20E+06 2.20E+06 1.55E+06 3.24E+06 S-Glutathionyl-L-cysteine 4.22E+04 2.07E+04 3.76E+04 2.75E+04 4.37E+04 2.74E+04 3.97E+04 2.15E+04 2.90E+04 9.65E+04 4.64E+04 0.00E+00 5.91E+04 1.11E+04 5.78E+04 1.93E+05 5.60E+04 2.66E+05 Dehydroascorbate 2.46E+05 2.50E+05 4.06E+05 3.98E+05 4.42E+05 3.38E+05 9.47E+05 4.97E+05 7.00E+05 2.90E+05 1.35E+05 1.55E+05 3.09E+05 1.64E+05 6.57E+04 7.22E+03 2.55E+04 3.28E+04 Dimethylglycine 1.90E+05 9.80E+04 1.24E+05 1.74E+05 2.15E+05 7.10E+04 1.85E+05 1.34E+05 9.76E+04 4.22E+05 2.06E+05 1.86E+05 3.01E+05 2.80E+05 2.40E+05 2.55E+04 1.16E+04 5.91E+04 N-Acetylneuraminate 3.47E+05 1.93E+05 1.17E+05 1.17E+05 1.59E+05 4.71E+04 9.26E+04 1.02E+05 5.79E+04 4.11E+05 4.46E+05 1.74E+05 2.15E+05 2.25E+05 1.92E+05 4.86E+04 4.07E+04 4.29E+04 UDP-N-acetyl-D-glucosamine 7.11E+05 1.03E+06 4.37E+05 8.65E+05 4.70E+05 7.80E+05 7.97E+05 6.82E+05 7.15E+05 1.82E+06 1.55E+06 1.67E+06 2.38E+06 2.18E+06 2.12E+06 1.47E+05 6.88E+04 9.15E+04 CMP-N-acetylneuraminate 4.01E+04 2.80E+04 3.28E+04 5.53E+04 3.95E+04 4.71E+04 4.06E+04 3.25E+04 3.40E+04 4.30E+04 2.47E+04 3.76E+04 5.98E+04 3.41E+04 5.46E+04 1.33E+05 4.16E+04 1.80E+05 trans-4-Hydroxy-L-proline 1.22E+04 9.07E+03 4.94E+03 4.76E+03 4.03E+03 3.70E+03 6.67E+03 6.62E+03 4.03E+03 8.45E+03 7.89E+03 7.33E+03 9.29E+03 1.60E+04 6.99E+03 1.71E+04 4.29E+03 7.76E+03 5-6-Dihydrouracil 1.26E+05 7.14E+04 4.96E+04 3.65E+04 7.37E+04 3.48E+04 3.95E+04 3.66E+04 1.73E+04 1.22E+05 1.06E+05 2.65E+04 6.50E+04 4.02E+04 4.05E+04 6.15E+03 6.03E+03 2.45E+04 L-Selenomethionine 1.17E+04 1.64E+04 1.27E+04 1.84E+04 1.51E+04 1.44E+04 1.51E+04 1.24E+04 1.95E+04 1.56E+04 9.09E+03 1.66E+04 1.70E+04 2.01E+04 9.29E+03 1.73E+04 1.75E+04 1.68E+04 Indole 1.09E+04 1.07E+04 7.47E+03 1.37E+04 1.15E+04 1.10E+04 1.20E+04 9.44E+03 1.19E+04 4.45E+03 2.15E+03 2.11E+03 2.04E+03 1.03E+03 1.43E+03 3.88E+02 1.20E+03 9.22E+02 3D-(3-5/4)-Trihydroxycyclohexane-1-2-dione 1.16E+04 6.87E+03 9.06E+03 5.88E+03 5.25E+03 4.22E+03 1.55E+04 8.24E+03 1.97E+04 1.29E+04 8.13E+03 1.57E+04 1.32E+04 9.32E+03 6.67E+03 3.24E+04 2.27E+04 3.63E+04 Glycerol 3-phosphate 2.03E+06 2.24E+06 6.03E+05 1.37E+06 1.32E+06 8.95E+05 1.00E+06 1.14E+06 1.30E+06 4.45E+06 4.43E+06 3.91E+06 3.07E+06 4.21E+06 1.97E+06 1.83E+05 2.76E+05 1.44E+05 FA(18:0) 1.98E+07 1.73E+07 1.42E+07 2.44E+07 2.29E+07 2.21E+07 1.38E+07 8.39E+06 1.38E+07 1.41E+07 8.22E+06 9.18E+06 1.28E+07 1.51E+07 5.50E+06 1.86E+07 1.36E+07 1.18E+07 FA(16:1) 3.96E+05 3.49E+05 4.66E+05 4.77E+05 4.05E+05 3.67E+05 4.87E+05 3.45E+05 6.03E+05 4.33E+05 3.17E+05 5.69E+05 7.11E+05 6.01E+05 2.35E+05 5.83E+05 5.10E+05 5.08E+05 FA(20:4) 6.86E+05 8.14E+05 5.05E+05 8.96E+05 7.06E+05 7.06E+05 8.90E+05 6.49E+05 8.05E+05 1.05E+06 7.18E+05 1.30E+06 7.92E+05 1.36E+06 6.27E+05 4.25E+05 3.16E+05 3.50E+05 FA(20:5) 2.06E+04 2.13E+04 1.31E+04 3.21E+04 1.42E+04 1.85E+04 2.52E+04 1.71E+04 1.94E+04 3.04E+04 2.51E+04 4.44E+04 3.99E+04 7.06E+04 2.84E+04 1.40E+04 1.32E+04 1.89E+04 FA(22:6) 8.25E+04 8.72E+04 4.25E+04 9.06E+04 6.13E+04 8.22E+04 6.72E+04 7.13E+04 6.96E+04 9.16E+04 7.73E+04 1.53E+05 4.46E+04 7.09E+04 5.49E+04 1.75E+04 1.13E+04 2.79E+04 FA(20:3) 1.65E+05 1.25E+05 1.20E+05 2.47E+05 1.76E+05 1.55E+05 2.10E+05 1.57E+05 1.74E+05 1.57E+05 1.57E+05 2.51E+05 3.06E+05 4.39E+05 1.14E+05 1.09E+05 1.28E+05 1.07E+05 FA(22:5) 1.41E+05 1.26E+05 6.13E+04 1.72E+05 8.79E+04 1.01E+05 1.01E+05 6.45E+04 6.68E+04 8.25E+04 7.99E+04 1.56E+05 4.66E+04 4.99E+04 4.01E+04 3.43E+04 1.83E+04 2.50E+04 Sphinganine 1-phosphate 2.43E+05 1.78E+05 1.47E+05 1.34E+05 4.20E+05 1.91E+05 2.37E+05 2.67E+05 7.83E+05 3.00E+05 3.24E+05 3.85E+05 2.57E+05 3.06E+05 2.17E+05 1.08E+05 9.44E+04 9.46E+04 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CmpdID Pathway m/z RT Polarity L-aspartate C00049 Amino acids 132.029 0.588 [M-H]- L-glutamate C00025 Amino acids 146.045 0.586 [M-H]- ATP C00002 Nucleotides 505.989 0.554 [M-H]- Cytidine C00475 Nucleotides 242.078 0.611 [M-H]- UMP C00105 Nucleotides 323.028 0.588 [M-H]- Uracil C00106 Nucleotides 111.019 0.603 [M-H]- ITP C00081 Nucleotides 506.969 0.587 [M-H]- IMP C00130 Nucleotides 347.040 0.595 [M-H]- Inosine C00294 Nucleotides 267.072 0.586 [M-H]- 4-Pyridoxate C00847 Nucleotides 182.043 0.634 [M-H]- UDP-glucose C00029 Nucleotides 565.048 0.547 [M-H]- Phosphate C00009 Phosphates 96.968 0.585 [M-H]- Diphosphate C00013 Phosphates 176.935 0.57 [M-H]- D-Glucose C00031 Glycolysis 179.055 0.619 [M-H]- D-Hexose-phosphate C02965 Glycolysis 259.022 0.575 [M-H]- Phosphoenolpyruvate C00074 Glycolysis 166.974 0.536 [M-H]- Pyruvate C00022 Glycolysis 87.007 0.609 [M-H]- Lactate C01432 Glycolysis 89.023 0.65 [M-H]- Ribose/Ribulose/Arabinose/Xylose/Xylulose C00121 Other sugars 149.045 0.585 [M-H]- Citrate C00158 TCA cycle 191.019 0.573 [M-H]- Succinate C00042 TCA cycle 117.018 0.582 [M-H]- Fumarate C00122 TCA cycle 115.002 0.567 [M-H]- Malate C00149 TCA cycle 133.013 0.569 [M-H]- 2-Hydroxyglutarate C02630 Alternative Carboxylic acids 147.028 0.565 [M-H]- 6-Phospho-D-gluconate C00345 Pentose Phosphate Pathway 275.017 0.557 [M-H]- Glutathione C00051 GSH homeostasis 306.078 0.609 [M-H]- Glutathione disulfide C00127 GSH homeostasis 611.143 0.55 [M-H]- 5-Oxoproline C01879 GSH homeostasis 128.034 0.597 [M-H]- S-Glutathionyl-L-cysteine C05526 GSH homeostasis 425.081 0.58 [M-H]- Dehydroascorbate C05422 GSH homeostasis 173.008 0.571 [M-H]- Dimethylglycine C01026 Serine biosynthesis and one-carbon metabolism 102.055 0.592 [M-H]- N-Acetylneuraminate C00270 Aminosugars 308.099 0.597 [M-H]- UDP-N-acetyl-D-glucosamine C00043 Aminosugars 606.074 0.55 [M-H]- CMP-N-acetylneuraminate C00128 Aminosugars 613.140 0.54 [M-H]- trans-4-Hydroxy-L-proline C01157 Arginine and proline metabolism 130.050 0.582 [M-H]- 5-6-Dihydrouracil C00429 Panthothenate metabolism 113.034 0.598 [M-H]- L-Selenomethionine C05335 Sulfur metabolism 195.991 0.684 [M-H]- Indole C00463 Indole and Tryptophan 116.049 1.686 [M-H]- 3D-(3-5/4)-Trihydroxycyclohexane-1-2-dione C04287 Inositol 159.029 0.6 [M-H]- Glycerol 3-phosphate C00093 Glycerophospholipid biosynthesis 171.005 0.585 [M-H]- FA(18:0) C01530 Saturated Fatty acids 283.264 3.461 [M-H]- FA(16:1) C08362 Monounsaturated Fatty Acids 253.217 2.613 [M-H]- FA(20:4) C00219 Poly-unsaturated Fatty Acids 303.233 2.557 [M-H]- FA(20:5) C06428 Poly-unsaturated Fatty Acids 301.217 2.419 [M-H]- FA(22:6) C06429 Poly-unsaturated Fatty Acids 327.233 2.466 [M-H]- FA(20:3) C03242 Essential fatty acids 305.248 2.774 [M-H]- FA(22:5) C16513 Essential fatty acids 329.250 2.593 [M-H]- Sphinganine 1-phosphate C01120 Sphingolipid biosynthesis 380.257 3.812 [M-H]- METABOLITES_END #END