#METABOLOMICS WORKBENCH msholola_20250923_135832 DATATRACK_ID:6477 STUDY_ID:ST004248 ANALYSIS_ID:AN007071 PROJECT_ID:PR002678 VERSION 1 CREATED_ON October 2, 2025, 1:53 pm #PROJECT PR:PROJECT_TITLE A multi-omics exploration of biological responses upon high carotenoid intake PR:PROJECT_SUMMARY Scope Despite decades of research, aspects of the bioactive properties of PR:PROJECT_SUMMARY carotenoids in vivo remain confounded by our limited understanding of their PR:PROJECT_SUMMARY metabolic fates. Unbiased, systems-level approaches can help capture global PR:PROJECT_SUMMARY metabolic shifts associated with carotenoid-rich diets. Here, we present an PR:PROJECT_SUMMARY exploratory multi-omics integration–combining carotenoid/apocarotenoid PR:PROJECT_SUMMARY concentrations, untargeted lipidomics, and transcriptomics–to elucidate the PR:PROJECT_SUMMARY biological effects of tomato juices differing in b-carotene and lycopene PR:PROJECT_SUMMARY content. Methods and results This study leverages blood samples from a PR:PROJECT_SUMMARY previously conducted 4-week controlled parallel arm clinical trial. Thirty-five PR:PROJECT_SUMMARY healthy adults on standardized background diets consumed daily 360 mL high PR:PROJECT_SUMMARY lycopene tomato juice (42.5 mg lycopene/d), high b-carotene tomato juice (30.4 PR:PROJECT_SUMMARY mg b-carotene/d), or a macronutrient-matched control. Plasma (apo)carotenoids PR:PROJECT_SUMMARY and lipid profiles pre- and post-intervention were assessed, along with whole PR:PROJECT_SUMMARY blood transcriptomes sequenced at week 4 only. Pairwise correlations and PR:PROJECT_SUMMARY multiblock sparse-PLS-DA were utilized for multi-omics integration. Gene-set PR:PROJECT_SUMMARY enrichment revealed carotenoid-specific modulation of transcripts related to PR:PROJECT_SUMMARY immune function. Thirteen lipidomic features were significantly altered after PR:PROJECT_SUMMARY b-carotene intake versus 2 upon high lycopene intake. Multi-omics integration PR:PROJECT_SUMMARY resolved treatment groups, visualizing the distinct molecular signatures altered PR:PROJECT_SUMMARY by two high carotenoid diets. This approach revealed subtle biological shifts PR:PROJECT_SUMMARY are largely impacted by b-apo-13-carotenone levels. We document its inverse PR:PROJECT_SUMMARY relationship with retinoic acid receptors and its downstream genes for the first PR:PROJECT_SUMMARY time in humans, consistent with in vitro reports that b-apo-13-carotenone is an PR:PROJECT_SUMMARY antagonist for retinoic acid signaling. Conclusion Multi-omics integration PR:PROJECT_SUMMARY uncovers testable, hypothesis-generating insights about carotenoid metabolism PR:PROJECT_SUMMARY and how metabolites may modulate molecular signaling pathways in humans. PR:INSTITUTE Ohio State University PR:DEPARTMENT Food Science & Technology PR:LABORATORY Cooperstone Lab PR:LAST_NAME Sholola PR:FIRST_NAME Maria PR:ADDRESS 2255 Kenny Rd PR:EMAIL sholola.1@osu.edu PR:PHONE 7086463488 #STUDY ST:STUDY_TITLE A multi-omics exploration of biological responses upon high carotenoid intake ST:STUDY_SUMMARY Scope Despite decades of research, aspects of the bioactive properties of ST:STUDY_SUMMARY carotenoids in vivo remain confounded by our limited understanding of their ST:STUDY_SUMMARY metabolic fates. Unbiased, systems-level approaches can help capture global ST:STUDY_SUMMARY metabolic shifts associated with carotenoid-rich diets. Here, we present an ST:STUDY_SUMMARY exploratory multi-omics integration–combining carotenoid/apocarotenoid ST:STUDY_SUMMARY concentrations, untargeted lipidomics, and transcriptomics–to elucidate the ST:STUDY_SUMMARY biological effects of tomato juices differing in b-carotene and lycopene ST:STUDY_SUMMARY content. Methods and results This study leverages blood samples from a ST:STUDY_SUMMARY previously conducted 4-week controlled parallel arm clinical trial. Thirty-five ST:STUDY_SUMMARY healthy adults on standardized background diets consumed daily 360 mL high ST:STUDY_SUMMARY lycopene tomato juice (42.5 mg lycopene/d), high b-carotene tomato juice (30.4 ST:STUDY_SUMMARY mg b-carotene/d), or a macronutrient-matched control. Plasma (apo)carotenoids ST:STUDY_SUMMARY and lipid profiles pre- and post-intervention were assessed, along with whole ST:STUDY_SUMMARY blood transcriptomes sequenced at week 4 only. Pairwise correlations and ST:STUDY_SUMMARY multiblock sparse-PLS-DA were utilized for multi-omics integration. Gene-set ST:STUDY_SUMMARY enrichment revealed carotenoid-specific modulation of transcripts related to ST:STUDY_SUMMARY immune function. Thirteen lipidomic features were significantly altered after ST:STUDY_SUMMARY b-carotene intake versus 2 upon high lycopene intake. Multi-omics integration ST:STUDY_SUMMARY resolved treatment groups, visualizing the distinct molecular signatures altered ST:STUDY_SUMMARY by two high carotenoid diets. This approach revealed subtle biological shifts ST:STUDY_SUMMARY are largely impacted by b-apo-13-carotenone levels. We document its inverse ST:STUDY_SUMMARY relationship with retinoic acid receptors and its downstream genes for the first ST:STUDY_SUMMARY time in humans, consistent with in vitro reports that b-apo-13-carotenone is an ST:STUDY_SUMMARY antagonist for retinoic acid signaling. Conclusion Multi-omics integration ST:STUDY_SUMMARY uncovers testable, hypothesis-generating insights about carotenoid metabolism ST:STUDY_SUMMARY and how metabolites may modulate molecular signaling pathways in humans. ST:INSTITUTE Ohio State University ST:DEPARTMENT Food Science & Technology ST:LABORATORY Cooperstone Lab ST:LAST_NAME Sholola ST:FIRST_NAME Maria ST:ADDRESS 2255 Kenny Rd ST:EMAIL sholola.1@osu.edu ST:PHONE 17086463488 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 35 ST:NUM_MALES 17 ST:NUM_FEMALES 18 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 21-75 SU:GENDER Male and female SU:HUMAN_INCLUSION_CRITERIA Subjects were required to have blood glucose <126 mg/dL and triglycerides <300 SU:HUMAN_INCLUSION_CRITERIA mg/dL SU:HUMAN_EXCLUSION_CRITERIA Subjects were excluded if any of the following were present: cancer, type 2 SU:HUMAN_EXCLUSION_CRITERIA diabetes, gastrointestinal surgeries or malabsorptive disorders, metabolic SU:HUMAN_EXCLUSION_CRITERIA disease, pregnancy or currently lactating, or use of blood thinning medications #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names 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RAW_FILE_NAME=5115-b3-lyc_c18neg_70.d SUBJECT_SAMPLE_FACTORS 5116 5116-b1-control_c18neg_6 Sample source:blood plasma | Timepoint:pre control RAW_FILE_NAME=5116-b1-control_c18neg_6.d SUBJECT_SAMPLE_FACTORS 5116 5116-b3-control_c18neg_20 Sample source:blood plasma | Timepoint:post control RAW_FILE_NAME=5116-b3-control_c18neg_20.d SUBJECT_SAMPLE_FACTORS 5117 5117-b1-lyc_c18neg_46 Sample source:blood plasma | Timepoint:pre red RAW_FILE_NAME=5117-b1-lyc_c18neg_46.d SUBJECT_SAMPLE_FACTORS 5117 5117-b3-lyc_c18neg_2 Sample source:blood plasma | Timepoint:post red RAW_FILE_NAME=5117-b3-lyc_c18neg_2.d SUBJECT_SAMPLE_FACTORS 5118 5118-b1-lyc_c18neg_57 Sample source:blood plasma | Timepoint:pre red RAW_FILE_NAME=5118-b1-lyc_c18neg_57.d SUBJECT_SAMPLE_FACTORS 5118 5118-b3-lyc_c18neg_9 Sample source:blood plasma | Timepoint:post red RAW_FILE_NAME=5118-b3-lyc_c18neg_9.d SUBJECT_SAMPLE_FACTORS 5119 5119-b1-control_c18neg_74 Sample source:blood plasma | Timepoint:pre control RAW_FILE_NAME=5119-b1-control_c18neg_74.d SUBJECT_SAMPLE_FACTORS 5119 5119-b3-control_c18neg_58 Sample source:blood plasma | Timepoint:post control RAW_FILE_NAME=5119-b3-control_c18neg_58.d SUBJECT_SAMPLE_FACTORS 5120 5120-b1-beta_c18neg_72 Sample source:blood plasma | Timepoint:pre beta RAW_FILE_NAME=5120-b1-beta_c18neg_72.d SUBJECT_SAMPLE_FACTORS 5120 5120-b3-beta_c18neg_54 Sample source:blood plasma | Timepoint:post beta RAW_FILE_NAME=5120-b3-beta_c18neg_54.d SUBJECT_SAMPLE_FACTORS 5121 5121-b1-beta_c18neg_69 Sample source:blood plasma | Timepoint:pre beta RAW_FILE_NAME=5121-b1-beta_c18neg_69.d SUBJECT_SAMPLE_FACTORS 5121 5121-b3-beta_c18neg_48 Sample source:blood plasma | Timepoint:post beta RAW_FILE_NAME=5121-b3-beta_c18neg_48.d SUBJECT_SAMPLE_FACTORS 5122 5122-b1-lyc_c18neg_14 Sample source:blood plasma | Timepoint:pre red RAW_FILE_NAME=5122-b1-lyc_c18neg_14.d SUBJECT_SAMPLE_FACTORS 5122 5122-b3-lyc_c18neg_75 Sample source:blood plasma | Timepoint:post red RAW_FILE_NAME=5122-b3-lyc_c18neg_75.d SUBJECT_SAMPLE_FACTORS 5123 5123-b1-control_c18neg_11 Sample source:blood plasma | Timepoint:pre control RAW_FILE_NAME=5123-b1-control_c18neg_11.d SUBJECT_SAMPLE_FACTORS 5123 5123-b3-control_c18neg_40 Sample source:blood plasma | Timepoint:post control RAW_FILE_NAME=5123-b3-control_c18neg_40.d SUBJECT_SAMPLE_FACTORS 5124 5124-b1-control_c18neg_45 Sample source:blood plasma | Timepoint:pre control RAW_FILE_NAME=5124-b1-control_c18neg_45.d SUBJECT_SAMPLE_FACTORS 5124 5124-b3-control_c18neg_68 Sample source:blood plasma | Timepoint:post control RAW_FILE_NAME=5124-b3-control_c18neg_68.d SUBJECT_SAMPLE_FACTORS 5125 5125-b1-control_c18neg_80 Sample source:blood plasma | Timepoint:pre control RAW_FILE_NAME=5125-b1-control_c18neg_80.d SUBJECT_SAMPLE_FACTORS 5125 5125-b3-control_c18neg_51 Sample source:blood plasma | Timepoint:post control RAW_FILE_NAME=5125-b3-control_c18neg_51.d SUBJECT_SAMPLE_FACTORS 5126 5126-b1-beta_c18neg_62 Sample source:blood plasma | Timepoint:pre beta RAW_FILE_NAME=5126-b1-beta_c18neg_62.d SUBJECT_SAMPLE_FACTORS 5126 5126-b3-beta_c18neg_24 Sample source:blood plasma | Timepoint:post beta RAW_FILE_NAME=5126-b3-beta_c18neg_24.d SUBJECT_SAMPLE_FACTORS 5127 5127-b1-control_c18neg_30 Sample source:blood plasma | Timepoint:pre control RAW_FILE_NAME=5127-b1-control_c18neg_30.d SUBJECT_SAMPLE_FACTORS 5127 5127-b3-control_c18neg_83 Sample source:blood plasma | Timepoint:post control RAW_FILE_NAME=5127-b3-control_c18neg_83.d SUBJECT_SAMPLE_FACTORS 5128 5128-b1-lyc_c18neg_8 Sample source:blood plasma | Timepoint:pre red RAW_FILE_NAME=5128-b1-lyc_c18neg_8.d SUBJECT_SAMPLE_FACTORS 5128 5128-b3-lyc_c18neg_60 Sample source:blood plasma | Timepoint:post red RAW_FILE_NAME=5128-b3-lyc_c18neg_60.d SUBJECT_SAMPLE_FACTORS 5129 5129-b1-lyc_c18neg_78 Sample source:blood plasma | Timepoint:pre red RAW_FILE_NAME=5129-b1-lyc_c18neg_78.d SUBJECT_SAMPLE_FACTORS 5129 5129-b3-lyc_c18neg_16 Sample source:blood plasma | Timepoint:post red RAW_FILE_NAME=5129-b3-lyc_c18neg_16.d SUBJECT_SAMPLE_FACTORS 5130 5130-b1-lyc_c18neg_17 Sample source:blood plasma | Timepoint:pre red RAW_FILE_NAME=5130-b1-lyc_c18neg_17.d SUBJECT_SAMPLE_FACTORS 5130 5130-b3-lyc_c18neg_50 Sample source:blood plasma | Timepoint:post red RAW_FILE_NAME=5130-b3-lyc_c18neg_50.d SUBJECT_SAMPLE_FACTORS 5131 5131-b1-beta_c18neg_38 Sample source:blood plasma | Timepoint:pre beta RAW_FILE_NAME=5131-b1-beta_c18neg_38.d SUBJECT_SAMPLE_FACTORS 5131 5131-b3-beta_c18neg_3 Sample source:blood plasma | Timepoint:post beta RAW_FILE_NAME=5131-b3-beta_c18neg_3.d SUBJECT_SAMPLE_FACTORS 5132 5132-b1-beta_c18neg_41 Sample source:blood plasma | Timepoint:pre beta RAW_FILE_NAME=5132-b1-beta_c18neg_41.d SUBJECT_SAMPLE_FACTORS 5132 5132-b3-beta_c18neg_52 Sample source:blood plasma | Timepoint:post beta RAW_FILE_NAME=5132-b3-beta_c18neg_52.d SUBJECT_SAMPLE_FACTORS 5133 5133-b1-control_c18neg_27 Sample source:blood plasma | Timepoint:pre control RAW_FILE_NAME=5133-b1-control_c18neg_27.d SUBJECT_SAMPLE_FACTORS 5133 5133-b3-control_c18neg_15 Sample source:blood plasma | Timepoint:post control RAW_FILE_NAME=5133-b3-control_c18neg_15.d SUBJECT_SAMPLE_FACTORS 5134 5134-b1-beta_c18neg_42 Sample source:blood plasma | Timepoint:pre beta RAW_FILE_NAME=5134-b1-beta_c18neg_42.d SUBJECT_SAMPLE_FACTORS 5134 5134-b3-beta_c18neg_64 Sample source:blood plasma | Timepoint:post beta RAW_FILE_NAME=5134-b3-beta_c18neg_64.d SUBJECT_SAMPLE_FACTORS 5135 5135-b1-lyc_c18neg_12 Sample source:blood plasma | Timepoint:pre red RAW_FILE_NAME=5135-b1-lyc_c18neg_12.d SUBJECT_SAMPLE_FACTORS 5135 5135-b3-lyc_c18neg_18 Sample source:blood plasma | Timepoint:post red RAW_FILE_NAME=5135-b3-lyc_c18neg_18.d SUBJECT_SAMPLE_FACTORS 5136 5136-b1-beta_c18neg_44 Sample source:blood plasma | Timepoint:pre beta RAW_FILE_NAME=5136-b1-beta_c18neg_44.d SUBJECT_SAMPLE_FACTORS 5136 5136-b3-beta_c18neg_77 Sample source:blood plasma | Timepoint:post beta RAW_FILE_NAME=5136-b3-beta_c18neg_77.d SUBJECT_SAMPLE_FACTORS QC1 qc1_c18neg_1 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc1_c18neg_1.d SUBJECT_SAMPLE_FACTORS QC1 qc1_c18pos_1 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc1_c18pos_1.d SUBJECT_SAMPLE_FACTORS QC2 qc2_c18neg_7 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc2_c18neg_7.d SUBJECT_SAMPLE_FACTORS QC2 qc2_c18pos_7 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc2_c18pos_7.d SUBJECT_SAMPLE_FACTORS QC3 qc3_c18neg_13 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc3_c18neg_13.d SUBJECT_SAMPLE_FACTORS QC3 qc3_c18pos_13 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc3_c18pos_13.d SUBJECT_SAMPLE_FACTORS QC4 qc4_c18neg_19 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc4_c18neg_19.d SUBJECT_SAMPLE_FACTORS QC4 qc4_c18pos_19 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc4_c18pos_19.d SUBJECT_SAMPLE_FACTORS QC5 qc5_c18neg_25 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc5_c18neg_25.d SUBJECT_SAMPLE_FACTORS QC5 qc5_c18pos_25 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc5_c18pos_25.d SUBJECT_SAMPLE_FACTORS QC6 qc6_c18neg_31 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc6_c18neg_31.d SUBJECT_SAMPLE_FACTORS QC6 qc6_c18pos_31 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc6_c18pos_31.d SUBJECT_SAMPLE_FACTORS QC7 qc7_c18neg_37 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc7_c18neg_37.d SUBJECT_SAMPLE_FACTORS QC7 qc7_c18pos_37 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc7_c18pos_37.d SUBJECT_SAMPLE_FACTORS QC8 qc8_c18neg_43 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc8_c18neg_43.d SUBJECT_SAMPLE_FACTORS QC8 qc8_c18pos_43 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc8_c18pos_43.d SUBJECT_SAMPLE_FACTORS QC9 qc9_c18neg_49 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc9_c18neg_49.d SUBJECT_SAMPLE_FACTORS QC9 qc9_c18pos_49 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc9_c18pos_49.d SUBJECT_SAMPLE_FACTORS QC10 qc10_c18neg_55 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc10_c18neg_55.d SUBJECT_SAMPLE_FACTORS QC10 qc10_c18pos_55 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc10_c18pos_55.d SUBJECT_SAMPLE_FACTORS QC11 qc11_c18neg_61 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc11_c18neg_61.d SUBJECT_SAMPLE_FACTORS QC11 qc11_c18pos_61 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc11_c18pos_61.d SUBJECT_SAMPLE_FACTORS QC12 qc12_c18neg_67 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc12_c18neg_67.d SUBJECT_SAMPLE_FACTORS QC12 qc12_c18pos_67 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc12_c18pos_67.d SUBJECT_SAMPLE_FACTORS QC13 qc13_c18neg_73 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc13_c18neg_73.d SUBJECT_SAMPLE_FACTORS QC13 qc13_c18pos_73 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc13_c18pos_73.d SUBJECT_SAMPLE_FACTORS QC14 qc14_c18neg_79 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc14_c18neg_79.d SUBJECT_SAMPLE_FACTORS QC14 qc14_c18pos_79 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc14_c18pos_79.d SUBJECT_SAMPLE_FACTORS QC15 qc15_c18neg_85 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc15_c18neg_85.d SUBJECT_SAMPLE_FACTORS QC15 qc15_c18pos_85 Sample source:blood plasma | Timepoint:pooled quality control RAW_FILE_NAME=qc15_c18pos_85.d #COLLECTION CO:COLLECTION_SUMMARY This study leveraged blood samples from a previously conducted parallel-arm CO:COLLECTION_SUMMARY clinical trial (NCT2550483). CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY The intervention was a high-lycopene (red) tomato juice or a high-β-carotene TR:TREATMENT_SUMMARY (orange) tomato juice, both derived from inbred tomato lines developed through TR:TREATMENT_SUMMARY conventional breeding. Thirty-five healthy adults on standardized background TR:TREATMENT_SUMMARY diets consumed daily 360 mL high lycopene tomato juice (42.5 mg lycopene/d), TR:TREATMENT_SUMMARY high b-carotene tomato juice (30.4 mg b-carotene/d), or a macronutrient-matched TR:TREATMENT_SUMMARY control. Plasma (apo)carotenoids and lipid profiles pre- and post-intervention TR:TREATMENT_SUMMARY were assessed, along with whole blood transcriptomes sequenced at week 4 only. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY In low light, blood plasma samples were thawed in cold water and vortexed to SP:SAMPLEPREP_SUMMARY ensure redistribution of lipids prior to extraction. Fifty microliters of thawed SP:SAMPLEPREP_SUMMARY plasma samples were transferred into centrifuge tubes, followed by an addition SP:SAMPLEPREP_SUMMARY of 250 microliters cold methanol with 10 mM ammonium formate to precipitate SP:SAMPLEPREP_SUMMARY proteins. Samples were then homogenized in a GenoGrinder 2010 (SPEX Sample Prep, SP:SAMPLEPREP_SUMMARY Metuchen, NJ, USA) for 2 min at 1400 RPM, and 250 microliters of butanol with 10 SP:SAMPLEPREP_SUMMARY mM ammonium formate was added to each sample. Extracts were centrifuged at SP:SAMPLEPREP_SUMMARY 13,000 x g for 10 min at 4 deg C. Process blanks were prepared with water SP:SAMPLEPREP_SUMMARY replacing the plasma and following the same steps while quality control (QC) SP:SAMPLEPREP_SUMMARY samples were made by pooling 50 microliters from each plasma sample at the end SP:SAMPLEPREP_SUMMARY of extraction. This method was adapted from Huynh and colleagues (doi: SP:SAMPLEPREP_SUMMARY 10.1016/j.chembiol.2018.10.008) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Agilent ZORBAX Eclipse Plus C18 (100 x 2.1mm,1.8um) CH:SOLVENT_A 50% water/30% acetonitrile/20% isopropanol; 10mM ammonium formate CH:SOLVENT_B 1% water/9% acetonitrile/90% isopropanol; 10mM ammonium formate CH:FLOW_GRADIENT A stepped-linear 16 min gradient was applied at 0.4 mL/min as described: held at CH:FLOW_GRADIENT 50% B for 2.5 min, quickly increased to 57% B over 0.1 min, increased linearly CH:FLOW_GRADIENT to 70% B until reaching 9 min, increased quickly to 93% B for 0.1 min and then CH:FLOW_GRADIENT increased to 96% B for 1.1 min. At 11.1 min, the gradient was held at 100% B CH:FLOW_GRADIENT until 12 min, decreased to 15% B for 0.2 min, and then held there for 3.8 min. CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 45 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6546 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS In negative ionization mode, all of the parameters were the same, except for the MS:MS_COMMENTS capillary voltage set to 3000 V. MS:MS_RESULTS_FILE ST004248_AN007071_Results.txt UNITS:Peak height Has m/z:Yes Has RT:Yes RT units:Minutes #END