#METABOLOMICS WORKBENCH Yuanye_Chi_20240730_073919 DATATRACK_ID:5050 STUDY_ID:ST003387 ANALYSIS_ID:AN005549 PROJECT_ID:PR002098 VERSION 1 CREATED_ON August 6, 2024, 10:19 pm #PROJECT PR:PROJECT_TITLE Annotation of Metabolites in Stable Isotope Tracing Untargeted Metabolomics via PR:PROJECT_TITLE Khipu-web PR:PROJECT_SUMMARY Stable isotope tracing is a crucial technique for understanding the metabolic PR:PROJECT_SUMMARY wiring of biological systems, determining meta-bolic flux through pathways of PR:PROJECT_SUMMARY interest, and detecting novel metabolites and pathways. Despite the potential PR:PROJECT_SUMMARY insights provid-ed by this technique, its application remains limited to a small PR:PROJECT_SUMMARY number of targeted molecules and pathways. Because previ-ous software tools PR:PROJECT_SUMMARY usually require chemical formulas to find relevant features, and the data are PR:PROJECT_SUMMARY highly complex, especially in untargeted metabolomics and when the reactions and PR:PROJECT_SUMMARY metabolites downstream the labeled substrates are poorly characterized. We PR:PROJECT_SUMMARY report here Khipu version 2 and its new user-friendly web application. New PR:PROJECT_SUMMARY functions are added to enhance analyzing stable isotope tracing data including PR:PROJECT_SUMMARY metrics that evaluate peak enrichment in labeled samples, scoring methods to PR:PROJECT_SUMMARY facilitate robust detection of intensity patterns and integrated natural PR:PROJECT_SUMMARY abundance correction. We demonstrate that this approach can be applied to PR:PROJECT_SUMMARY untargeted metabolomics to systematically extract isotope-labeled compounds and PR:PROJECT_SUMMARY annotate the unidentified me-tabolites. PR:INSTITUTE The Jackson Laboratory for Genomic Medicine PR:LABORATORY Shuzhao Li Lab PR:LAST_NAME Chi PR:FIRST_NAME Yuanye PR:ADDRESS 10 Discovery Dr, Farmington, CT PR:EMAIL yuanye.chi@jax.org PR:PHONE 3395456866 #STUDY ST:STUDY_TITLE Annotation of Metabolites in Stable Isotope Tracing Untargeted Metabolomics via ST:STUDY_TITLE Khipu-web ST:STUDY_SUMMARY Stable isotope tracing is a crucial technique for understanding the metabolic ST:STUDY_SUMMARY wiring of biological systems, determining meta-bolic flux through pathways of ST:STUDY_SUMMARY interest, and detecting novel metabolites and pathways. Despite the potential ST:STUDY_SUMMARY insights provid-ed by this technique, its application remains limited to a small ST:STUDY_SUMMARY number of targeted molecules and pathways. Because previ-ous software tools ST:STUDY_SUMMARY usually require chemical formulas to find relevant features, and the data are ST:STUDY_SUMMARY highly complex, especially in untargeted metabolomics and when the reactions and ST:STUDY_SUMMARY metabolites downstream the labeled substrates are poorly characterized. We ST:STUDY_SUMMARY report here Khipu version 2 and its new user-friendly web application. New ST:STUDY_SUMMARY functions are added to enhance analyzing stable isotope tracing data including ST:STUDY_SUMMARY metrics that evaluate peak enrichment in labeled samples, scoring methods to ST:STUDY_SUMMARY facilitate robust detection of intensity patterns and integrated natural ST:STUDY_SUMMARY abundance correction. We demonstrate that this approach can be applied to ST:STUDY_SUMMARY untargeted metabolomics to systematically extract isotope-labeled compounds and ST:STUDY_SUMMARY annotate the unidentified me-tabolites. ST:INSTITUTE The Jackson Laboratory for Genomic Medicine ST:LAST_NAME Chi ST:FIRST_NAME Yuanye ST:ADDRESS 10 Discovery Dr, Farmington, CT ST:EMAIL yuanye.chi@jax.org ST:PHONE 3395456866 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens #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 - 293T_Sample1_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample1_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample1_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample1_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample2_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample2_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample2_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample2_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample3_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample3_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample3_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample3_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample4_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample4_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample4_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample4_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample5_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample5_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample5_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample5_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample6_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample6_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample6_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample6_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample7_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample7_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample7_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample7_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample8_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:ctrl RAW_FILE_NAME(File Name)=293T_Sample8_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample8_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:ctrl RAW_FILE_NAME(File Name)=293T_Sample8_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample9_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample9_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample9_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample9_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample10_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample10_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample10_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample10_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample11_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample11_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample11_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample11_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample12_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample12_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample12_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample12_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample13_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample13_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample13_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample13_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample14_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample14_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample14_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample14_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample15_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample15_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample15_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample15_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample16_HILICneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample16_HILICneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample16_RPpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample16_RPpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample1_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample1_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample1_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample1_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample2_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample2_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample2_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample2_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample3_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample3_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample3_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample3_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample4_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample4_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample4_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample4_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample5_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample5_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample5_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample5_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample6_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample6_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample6_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample6_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample7_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample7_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample7_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:6hr RAW_FILE_NAME(File Name)=293T_Sample7_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample8_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:ctrl RAW_FILE_NAME(File Name)=293T_Sample8_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample8_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:ctrl RAW_FILE_NAME(File Name)=293T_Sample8_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample9_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample9_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample9_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:12hr RAW_FILE_NAME(File Name)=293T_Sample9_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample10_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample10_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample10_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample10_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample11_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample11_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample11_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample11_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample12_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample12_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample12_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample12_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample13_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample13_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample13_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample13_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample14_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample14_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample14_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample14_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample15_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample15_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample15_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample15_RPneg.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample16_HILICpos Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample16_HILICpos.mzML SUBJECT_SAMPLE_FACTORS - 293T_Sample16_RPneg Sample source:human embryonic kidney (HEK) 293T cells | Label Time Point:1hr RAW_FILE_NAME(File Name)=293T_Sample16_RPneg.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_009 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_009.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_010 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_010.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_011 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_011.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_012 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_012.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_013 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_013.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_014 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_014.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_015 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_015.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_016 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_016.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_017 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_017.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_018 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_018.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_019 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_019.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_020 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_020.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_021 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_021.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_022 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_022.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_023 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_023.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_024 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_024.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_025 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_025.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_026 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_026.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_027 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_027.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_028 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_028.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_031 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_031.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_032 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_032.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_033 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_033.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_034 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_034.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_035 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_035.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_036 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_036.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_037 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_037.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_038 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_038.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_039 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_039.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_040 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_040.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_041 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_041.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_042 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240318_042.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_043 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_043.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_044 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_044.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_045 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_045.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_046 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_046.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_047 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_047.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_048 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_048.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_049 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_049.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_050 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_050.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_053 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_053.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_054 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240318_054.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_055 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_055.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_056 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_056.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_057 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_057.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_058 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240318_058.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_059 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_059.mzML SUBJECT_SAMPLE_FACTORS - MT_20240318_060 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240318_060.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_009 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_009.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_010 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_010.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_011 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_011.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_012 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_012.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_013 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_013.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_014 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_014.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_015 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_015.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_016 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_016.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_017 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_017.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_018 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_018.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_019 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_019.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_020 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_020.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_021 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_021.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_022 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_022.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_023 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_023.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_024 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_024.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_025 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_025.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_026 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_026.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_027 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_027.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_028 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_028.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_031 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_031.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_032 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_032.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_033 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_033.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_034 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_034.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_035 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_035.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_036 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_036.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_037 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_037.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_038 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_038.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_039 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_039.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_040 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_040.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_041 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_041.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_042 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:6hr RAW_FILE_NAME(File Name)=MT_20240319_042.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_043 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_043.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_044 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_044.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_045 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_045.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_046 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_046.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_047 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_047.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_048 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_048.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_049 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_049.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_050 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_050.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_053 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_053.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_054 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:12hr RAW_FILE_NAME(File Name)=MT_20240319_054.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_055 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_055.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_056 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_056.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_057 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_057.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_058 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:ctrl RAW_FILE_NAME(File Name)=MT_20240319_058.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_059 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_059.mzML SUBJECT_SAMPLE_FACTORS - MT_20240319_060 Sample source:acute myeloid leukemia (AML) cell | Label Time Point:2hr RAW_FILE_NAME(File Name)=MT_20240319_060.mzML #COLLECTION CO:COLLECTION_SUMMARY Culturing of HEK 293T Cells HEK 293T cells were purchased from ATCC (CRL-3216, CO:COLLECTION_SUMMARY Manassas, Virginia, USA) and cultured using a modification of methods published CO:COLLECTION_SUMMARY by Lu et al. 2023 2. Cell culture media was prepared by mixing 50mL of fetal CO:COLLECTION_SUMMARY bovine serum (FBS, A3840101, Thermo Fisher Scientific) with 5mL of a 5000U/mL CO:COLLECTION_SUMMARY penicillin/streptomycin solution (15070063, Thermo Fisher Scientific) to 450 mL CO:COLLECTION_SUMMARY of high glucose DMEM (11965092, Thermo Fisher Scientific). Frozen culture media CO:COLLECTION_SUMMARY consisted of 60% culture media, 30% FBS and 10% DMSO prepared in 15-mL tubes and CO:COLLECTION_SUMMARY stored at -20°C. Cells were kept in liquid nitrogen prior to recovery (day 1) CO:COLLECTION_SUMMARY and were thawed within 1 minute in an incubator pre-warmed to 37°C. 1mL of CO:COLLECTION_SUMMARY culture media was absorbed in a 5-mL pipette then used to transfer all cell CO:COLLECTION_SUMMARY suspensions to a new 15mL tube which was then centrifuged at 300g for 4 minutes CO:COLLECTION_SUMMARY at room temperature. The supernatant was discarded and 1mL of media was used to CO:COLLECTION_SUMMARY then re-suspend the cells. The cell suspension was then transferred to a T25 CO:COLLECTION_SUMMARY plate containing 4mL of media which was then incubated at 37C and 5% CO2. After CO:COLLECTION_SUMMARY overnight incubation, on day 2, cells were checked for confluency via CO:COLLECTION_SUMMARY micrograph. At approximately 80 to 90% confluency, cells were washed with 2mL CO:COLLECTION_SUMMARY PBS (J61196-AP, Thermo Fisher Scientific) and the wash discarded without CO:COLLECTION_SUMMARY disturbing adhered cells. 0.5mL of trypsin buffer (1 ×, sterile; CO:COLLECTION_SUMMARY sterile-filtered, BioReagent, suitable for cell culture, 0.5 g porcine trypsin CO:COLLECTION_SUMMARY and 0.2 g EDTA in 100 mL, 4Na per liter of Hanks′ Balanced Salt Solution with CO:COLLECTION_SUMMARY phenol red) was added and then incubated at room temperature or at 37C depending CO:COLLECTION_SUMMARY on the amount of adherence for 5 minutes. 1.5mL of culture media was then added CO:COLLECTION_SUMMARY to resuspend the cells and then transferred to a 15mL tube. After centrifuging CO:COLLECTION_SUMMARY at 300g for 4 minutes at room temperature, the supernatant was discarded and the CO:COLLECTION_SUMMARY cells resuspended in 1mL of culture media prior to transfer to a 10-cm plate CO:COLLECTION_SUMMARY containing 9mL of culture media. Samples were incubated overnight at 37C and CO:COLLECTION_SUMMARY 5% CO2. This process was repeated on day 3 and 4 with the 10-cm plate and CO:COLLECTION_SUMMARY increased resuspension volumes, 3mL on day 3 and 7 mL of frozen media on day 4. CO:COLLECTION_SUMMARY At the end of day 4, one plate was retained for the isotope labeling experiments CO:COLLECTION_SUMMARY while the remaining was was transferred to frozen vials 1mL at a time for a CO:COLLECTION_SUMMARY target density of 10^6 cells/mL that were then stored at -80C for the CO:COLLECTION_SUMMARY entirety of day 5 prior to liquid nitrogen storage on day 6. Culturing of THP1 CO:COLLECTION_SUMMARY and MOLM 13 cells Two acute myeloid leukemia (AML) cell lines, THP1 (ACC 16) and CO:COLLECTION_SUMMARY MOLM13 (ACC 554), were obtained from DSMZ and cultured using a modification of CO:COLLECTION_SUMMARY methods from Wang et al. 2023 1. Fresh culture media was prepared by mixing 50mL CO:COLLECTION_SUMMARY of fetal bovine serum (FBS) (Corning, 35-011-CV), 5mL of a 5000U/mL CO:COLLECTION_SUMMARY penicillin/streptomycin solution (Corning, 30-009-CI), 5mL of glutamax (Gibco, CO:COLLECTION_SUMMARY 35-050-061) to 500 mL of RPMI-1640 media (Corning, 10-040-CV). MOLM-13 cells CO:COLLECTION_SUMMARY were obtained in a frozen cryovial and stored in liquid nitrogen prior to CO:COLLECTION_SUMMARY recovery (day 1) and were thawed within 1 minute in an incubator pre-warmed to CO:COLLECTION_SUMMARY 37C. 1mL of culture media was absorbed in a 5-mL pipette then transfer all CO:COLLECTION_SUMMARY cell suspensions to a new 15mL tube containing 9mL of 1xPBS, which was then CO:COLLECTION_SUMMARY centrifuged at 400g for 5 minutes at room temperature. The supernatant was CO:COLLECTION_SUMMARY discarded and 1mL of media was used to then re-suspend the cells. The cell CO:COLLECTION_SUMMARY suspension was then transferred to a T25 plate containing 4mL of media which was CO:COLLECTION_SUMMARY then incubated at 37C and 5% CO2. After overnight incubation, on day 2, cells CO:COLLECTION_SUMMARY were checked for confluency via micrograph. At approximately 80 to 90% CO:COLLECTION_SUMMARY confluency, the cells were centrifuged at 400g for 5 minutes. The cells were CO:COLLECTION_SUMMARY resuspended with 2mL of media and then split into two 10-cm disc containing 9mL CO:COLLECTION_SUMMARY of culture media. Culture discs were incubated overnight at 37C and 5% CO2. CO:COLLECTION_SUMMARY THP1 was obtained in T25 plate cultured in 10mL culture media. The cell CO:COLLECTION_SUMMARY suspension was then transferred to a T75 plate adding 10mL of media which was CO:COLLECTION_SUMMARY then incubated at 37C and 5% CO2. At approximately 80 to 90% confluency, the CO:COLLECTION_SUMMARY cells were centrifuged at 400g for 5 minutes and resuspended with 5mL of culture CO:COLLECTION_SUMMARY media. For subsequent culture, 1mL of cell suspension was added in each 10-cm CO:COLLECTION_SUMMARY disc and 4mL of media was added. This process was repeated for both cells to CO:COLLECTION_SUMMARY have enough culture disc for subsequent experiment and storage. For storage, CO:COLLECTION_SUMMARY frozen culture media consisted of 60% culture media, 30% FBS and 10% DMSO CO:COLLECTION_SUMMARY prepared in 15-mL tubes and stored at -20C. Ten 10-cm disc of THP1 and five CO:COLLECTION_SUMMARY 10-cm disc for MOLM13 were retained for isotope tracing experiment and the CO:COLLECTION_SUMMARY remaining was transferred to frozen vials 1mL at a time for a target density of CO:COLLECTION_SUMMARY 3*106 cells/mL that were then stored at -80C for 24 hour prior to liquid CO:COLLECTION_SUMMARY nitrogen storage. CO:SAMPLE_TYPE iPSC cells #TREATMENT TR:TREATMENT_SUMMARY Isotope Labeling of HEK 293T Cells First, a 10-cm plate of 293T cells was TR:TREATMENT_SUMMARY suspended with 7.2mL of culture media and four 6-well plates were prepared in TR:TREATMENT_SUMMARY which 3 plates contain 2.5mL of culture media in 4 wells while the remaining TR:TREATMENT_SUMMARY plate contains 2.5mL of culture media in all six wells. 200µL of cell TR:TREATMENT_SUMMARY suspension was added to each media-containing well and then incubated at 37C TR:TREATMENT_SUMMARY and 5% CO2 overnight. In isotope labeling experiment, culture media was prepared TR:TREATMENT_SUMMARY by mixing 50mL of dialyzed fetal bovine serum (A3382001) with 5mL of a 5000U/mL TR:TREATMENT_SUMMARY penicillin/streptomycin solution to 450 mL of glucose- and glutamine-free DMEM TR:TREATMENT_SUMMARY (A1443001). An isotope labeled culture media was created by mixing TR:TREATMENT_SUMMARY [U-13C6]-D-Glucose (CLM-1396, Cambridge Isotope Laboratories) and TR:TREATMENT_SUMMARY 13C5-L-Glutamine (CLM-1822-H-PK, Cambridge Isotope Laboratories) along with an TR:TREATMENT_SUMMARY appropriate amount of unlabeled glucose and glutamine (G7021 and G8540, TR:TREATMENT_SUMMARY Millipore Sigma, Sigma Aldrich) to 25mL of culture media to yield a 12.5mM TR:TREATMENT_SUMMARY [U-13C6]-D-Glucose, 2mM 13C5L-Glutamine, 12.5mM D-Glucose, and 2mM L-glutamine TR:TREATMENT_SUMMARY concentration. 13mLs of a control unlabeled media was created with a TR:TREATMENT_SUMMARY concentration of 25mM for unlabeled glucose and 4 mM for unlabeled glutamine. TR:TREATMENT_SUMMARY For two of the fully populated 6-well plates, the culture media was replaced TR:TREATMENT_SUMMARY with 2mL of the labeled media while the culture media of the remaining 6 wells TR:TREATMENT_SUMMARY were replaced with control media and then incubated at 37C and 5% CO2. At 2h, TR:TREATMENT_SUMMARY 6h, and 12h, cells were harvested from each labeled well while the control was TR:TREATMENT_SUMMARY harvested at 6h. Harvesting consisted of aspirating 1 mL of culture medium to a TR:TREATMENT_SUMMARY new 1.5 mL tube and centrifuging at 300g for 5 minutes at room temperature. TR:TREATMENT_SUMMARY After centrifugation, 500µL of supernatant was transferred to a new 1.5mL tube TR:TREATMENT_SUMMARY and stored at -80C for future analysis. The remaining supernatant was TR:TREATMENT_SUMMARY discarded. Cell pellets were harvested by first pipetting any remaining media TR:TREATMENT_SUMMARY out of the well without disturbing the cells. The well was then washed with 2mL TR:TREATMENT_SUMMARY of 1x PBS, and the wash volume aspirated before adding an additional 2mL of 1x TR:TREATMENT_SUMMARY PBS. Cells were then scraped gently with a cell lifter and then transferred to a TR:TREATMENT_SUMMARY 15mL tube containing 2mL of 1x PBS. These tubes were then centrifuged at 300g TR:TREATMENT_SUMMARY for 5 minutes at room temperature and the supernatant discarded. The cell pellet TR:TREATMENT_SUMMARY was resuspended with 1mL of 1x PBS and then counted with a hemocytometer as TR:TREATMENT_SUMMARY previously described. Cell pellets were then transferred with 1mL of 1x PBS to a TR:TREATMENT_SUMMARY 1.5mL tube that were then centrifuged at 300g for 5 minutes at room temperature. TR:TREATMENT_SUMMARY The supernatant was then discarded, and the cell pellets stored at -80C for TR:TREATMENT_SUMMARY future use. Culturing of THP1 and MOLM 13 cells Two acute myeloid leukemia (AML) TR:TREATMENT_SUMMARY cell lines, THP1 (ACC 16) and MOLM13 (ACC 554), were obtained from DSMZ and TR:TREATMENT_SUMMARY cultured using a modification of methods from Wang et al. 2023 1. Fresh culture TR:TREATMENT_SUMMARY media was prepared by mixing 50mL of fetal bovine serum (FBS) (Corning, TR:TREATMENT_SUMMARY 35-011-CV), 5mL of a 5000U/mL penicillin/streptomycin solution (Corning, TR:TREATMENT_SUMMARY 30-009-CI), 5mL of glutamax (Gibco, 35-050-061) to 500 mL of RPMI-1640 media TR:TREATMENT_SUMMARY (Corning, 10-040-CV). MOLM-13 cells were obtained in a frozen cryovial and TR:TREATMENT_SUMMARY stored in liquid nitrogen prior to recovery (day 1) and were thawed within 1 TR:TREATMENT_SUMMARY minute in an incubator pre-warmed to 37C. 1mL of culture media was absorbed TR:TREATMENT_SUMMARY in a 5-mL pipette then transfer all cell suspensions to a new 15mL tube TR:TREATMENT_SUMMARY containing 9mL of 1xPBS, which was then centrifuged at 400g for 5 minutes at TR:TREATMENT_SUMMARY room temperature. The supernatant was discarded and 1mL of media was used to TR:TREATMENT_SUMMARY then re-suspend the cells. The cell suspension was then transferred to a T25 TR:TREATMENT_SUMMARY plate containing 4mL of media which was then incubated at 37C and 5% CO2. TR:TREATMENT_SUMMARY After overnight incubation, on day 2, cells were checked for confluency via TR:TREATMENT_SUMMARY micrograph. At approximately 80 to 90% confluency, the cells were centrifuged at TR:TREATMENT_SUMMARY 400g for 5 minutes. The cells were resuspended with 2mL of media and then split TR:TREATMENT_SUMMARY into two 10-cm disc containing 9mL of culture media. Culture discs were TR:TREATMENT_SUMMARY incubated overnight at 37C and 5% CO2. THP1 was obtained in T25 plate TR:TREATMENT_SUMMARY cultured in 10mL culture media. The cell suspension was then transferred to a TR:TREATMENT_SUMMARY T75 plate adding 10mL of media which was then incubated at 37C and 5% CO2. At TR:TREATMENT_SUMMARY approximately 80 to 90% confluency, the cells were centrifuged at 400g for 5 TR:TREATMENT_SUMMARY minutes and resuspended with 5mL of culture media. For subsequent culture, 1mL TR:TREATMENT_SUMMARY of cell suspension was added in each 10-cm disc and 4mL of media was added. This TR:TREATMENT_SUMMARY process was repeated for both cells to have enough culture disc for subsequent TR:TREATMENT_SUMMARY experiment and storage. For storage, frozen culture media consisted of 60% TR:TREATMENT_SUMMARY culture media, 30% FBS and 10% DMSO prepared in 15-mL tubes and stored at TR:TREATMENT_SUMMARY -20C. Ten 10-cm disc of THP1 and five 10-cm disc for MOLM13 were retained for TR:TREATMENT_SUMMARY isotope tracing experiment and the remaining was transferred to frozen vials 1mL TR:TREATMENT_SUMMARY at a time for a target density of 3*106 cells/mL that were then stored at TR:TREATMENT_SUMMARY -80C for 24 hour prior to liquid nitrogen storage. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Single Phase Cell Pellet Extraction Pellets were transferred to a 1.5 mL SP:SAMPLEPREP_SUMMARY Eppendorf tube for extraction. An empty tube was also extracted to serve as a SP:SAMPLEPREP_SUMMARY process blank. To each cell pellet, 100µL of ice cold (4ºC) 80% MeOH in water SP:SAMPLEPREP_SUMMARY was added and 4µL of an IS spike-in solution was added for the AML cell SP:SAMPLEPREP_SUMMARY samples. This IS solution is a stock solution prepared by mixing 0.12 mL of 1 M SP:SAMPLEPREP_SUMMARY D-Glucose-13C6, 0.2 mL of 5 mM Caffeine-3-methyl-13C1, 0.6 mL of 10 mM SP:SAMPLEPREP_SUMMARY L-Methionine-13C5, 0.6 mL of 20 mM L-Glutamic acid-13C5, 0.8 mL of 10 mM SP:SAMPLEPREP_SUMMARY Uracil-15N2, 4 mL of 2 mM L-Tyrosine-15N1 and 3.68 mL water in 15-mL tube. SP:SAMPLEPREP_SUMMARY Samples were then vortexed vigorously for 30 seconds and then subjected to 3 SP:SAMPLEPREP_SUMMARY freeze-thaw cycles. Each freeze-thaw cycle consisted of placing the sample in SP:SAMPLEPREP_SUMMARY liquid nitrogen for 30 seconds, then thawing in an ice bath and sonicating for 3 SP:SAMPLEPREP_SUMMARY minutes. The sample was then vortexed for 10 additional seconds prior to SP:SAMPLEPREP_SUMMARY centrifuging the sample at 20,817g for 20 minutes at 4ºC. 100µL of the SP:SAMPLEPREP_SUMMARY resulting supernatant was then transferred to a new 1.5mL eppendorf tube. For SP:SAMPLEPREP_SUMMARY the AML samples, 10µL of each study sample, labeled and unlabelled, was SP:SAMPLEPREP_SUMMARY aliquoted to a new 1.5mL eppendorf tube for the creation of a pooled QC sample. SP:SAMPLEPREP_SUMMARY For subsequent DDA experiments, a new unlabeled pooled sample was created in a SP:SAMPLEPREP_SUMMARY similar fashion using 20µL from each of the unlabelled samples only. Samples SP:SAMPLEPREP_SUMMARY from both cell lines, THP1 and MOLM13, were used for both pooled samples. For SP:SAMPLEPREP_SUMMARY 293T cells, the same procedure was used to create the pooled samples for both QC SP:SAMPLEPREP_SUMMARY and unlabeled DDA; however, a volume of 10µL was aliquoted each time. For 293T SP:SAMPLEPREP_SUMMARY cells 30µL of the supernatant was transferred to an autosampler vial while for SP:SAMPLEPREP_SUMMARY AML cells 60µL was transferred. For all samples, 3µL was injected for each SP:SAMPLEPREP_SUMMARY LC-MS method, both full scan and DDA. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Accucore HILIC (100 x 2.1mm,2.6um) CH:SOLVENT_A 95% acetonitrile/5% water; 10 mM ammonium acetate;0.1% acetic acid CH:SOLVENT_B 50% acetonitrile/50% water; 10 mM ammonium acetate; 0.1% acetic acid CH:FLOW_GRADIENT 0-0.2 min: 0% B, 0.20-8.75 min: 98% B, and 11.25 min for cleaning and CH:FLOW_GRADIENT equilibration of column CH:FLOW_RATE 0.55 ml/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS mass range, 100-1700 m/z for RP data acquisition and 60-1000 for HILIC data MS:MS_COMMENTS acquisition; spray voltage, 3200 V (ESI+), 2800 V (ESI-); sheath gas, 45 Arb; MS:MS_COMMENTS auxiliary gas, 20 Arb; sweep gas, 1 Arb; ion transfer tube temperature, 325 °C; MS:MS_COMMENTS vaporizer temperature, 325 °C; full scan mass resolution, 60,000 (MS1); MS:MS_COMMENTS normalized AGC target (%), 25; maximum injection time, 100 ms. isolation window MS:MS_COMMENTS (m/z), 1.2; stepped HCD collision energy (%), 20,40,80; dd-MS/MS resolution, MS:MS_COMMENTS 30,000; normalized AGC target (%), 20; maximum injection time (ms), 54; micro MS:MS_COMMENTS scan, 1; cycle time (sec), 1.2. MS:MS_RESULTS_FILE ST003387_AN005549_Results.txt UNITS:peak intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END