#METABOLOMICS WORKBENCH FJR_20241019_012821 DATATRACK_ID:5295 STUDY_ID:ST003652 ANALYSIS_ID:AN005999 PROJECT_ID:PR002262 VERSION 1 CREATED_ON January 7, 2025, 5:13 am #PROJECT PR:PROJECT_TITLE Multiplatform Metabolomic Profiling of the Unilateral Ureteral Obstruction PR:PROJECT_TITLE Murine Model of CKD PR:PROJECT_SUMMARY In chronic kidney disease (CKD) research, animal models provide invaluable PR:PROJECT_SUMMARY insights into the disease’s etiopathogenesis and progression, particularly PR:PROJECT_SUMMARY through the evaluation of renal tissue. The unilateral ureteral obstruction PR:PROJECT_SUMMARY (UUO) rodent model stands out for its widespread use in CKD studies, due to its PR:PROJECT_SUMMARY advantages to generate renal fibrosis and accelerated mimicry of obstructive PR:PROJECT_SUMMARY nephropathy in humans. Despite its extensive use, the molecular underpinnings PR:PROJECT_SUMMARY driving kidney disease progression remain incompletely understood. Given the PR:PROJECT_SUMMARY crucial interplay between metabolism and fibrosis in CKD, a thorough examination PR:PROJECT_SUMMARY of the UUO renal tissue through metabolomics is required. Untargeted PR:PROJECT_SUMMARY multiplatform analysis enables a comprehensive measurement of the sample PR:PROJECT_SUMMARY metabolic profile, ensuring a maximum coverage of metabolite diversity to yield PR:PROJECT_SUMMARY extensive insights into the metabolism of this renal injury model. Therefore, in PR:PROJECT_SUMMARY this study, murine kidney tissue from the UUO model underwent analysis using PR:PROJECT_SUMMARY three separation techniques—liquid chromatography (LC), gas chromatography PR:PROJECT_SUMMARY (GC), and capillary electrophoresis (CE)—coupled with mass spectrometry (MS). PR:PROJECT_SUMMARY The findings reveal metabolic changes associated with tubulointerstitial PR:PROJECT_SUMMARY fibrosis, impacting essential pathways such as the TCA cycle, urea cycle, PR:PROJECT_SUMMARY polyamine metabolism, amino acids, one-carbon metabolism, purine catabolism, and PR:PROJECT_SUMMARY NAD+ synthesis, among others. Furthermore, fibrosis significantly influences the PR:PROJECT_SUMMARY renal tissue's lipidomic profile, characterized by a general decrease in most PR:PROJECT_SUMMARY lipid classes and an increase in glycerophospholipids with ether substituents, PR:PROJECT_SUMMARY hexosylceramides, and cholesterol esters compared to the control. These results PR:PROJECT_SUMMARY underscore the relevance of the untargeted multiplatform approach to obtain a PR:PROJECT_SUMMARY comprehensive overview of the alterations within the renal metabolic map, paving PR:PROJECT_SUMMARY the way for further exploration of the molecular mechanisms underlying CKD. PR:INSTITUTE Universidad CEU San Pablo PR:LAST_NAME Rupérez PR:FIRST_NAME Francisco PR:ADDRESS Campus Montepríncipe. Ctra. M-501 km 0 PR:EMAIL ruperez@ceu.es PR:PHONE 913724753 #STUDY ST:STUDY_TITLE Multiplatform Metabolomic Profiling of the Unilateral Ureteral Obstruction ST:STUDY_TITLE Murine Model of CKD ST:STUDY_SUMMARY In chronic kidney disease (CKD) research, animal models provide invaluable ST:STUDY_SUMMARY insights into the disease’s etiopathogenesis and progression, particularly ST:STUDY_SUMMARY through the evaluation of renal tissue. The unilateral ureteral obstruction ST:STUDY_SUMMARY (UUO) rodent model stands out for its widespread use in CKD studies, due to its ST:STUDY_SUMMARY advantages to generate renal fibrosis and accelerated mimicry of obstructive ST:STUDY_SUMMARY nephropathy in humans. Despite its extensive use, the molecular underpinnings ST:STUDY_SUMMARY driving kidney disease progression remain incompletely understood. Given the ST:STUDY_SUMMARY crucial interplay between metabolism and fibrosis in CKD, a thorough examination ST:STUDY_SUMMARY of the UUO renal tissue through metabolomics is required. Untargeted ST:STUDY_SUMMARY multiplatform analysis enables a comprehensive measurement of the sample ST:STUDY_SUMMARY metabolic profile, ensuring a maximum coverage of metabolite diversity to yield ST:STUDY_SUMMARY extensive insights into the metabolism of this renal injury model. Therefore, in ST:STUDY_SUMMARY this study, murine kidney tissue from the UUO model underwent analysis using ST:STUDY_SUMMARY three separation techniques—liquid chromatography (LC), gas chromatography ST:STUDY_SUMMARY (GC), and capillary electrophoresis (CE)—coupled with mass spectrometry (MS). ST:STUDY_SUMMARY The findings reveal metabolic changes associated with tubulointerstitial ST:STUDY_SUMMARY fibrosis, impacting essential pathways such as the TCA cycle, urea cycle, ST:STUDY_SUMMARY polyamine metabolism, amino acids, one-carbon metabolism, purine catabolism, and ST:STUDY_SUMMARY NAD+ synthesis, among others. Furthermore, fibrosis significantly influences the ST:STUDY_SUMMARY renal tissue's lipidomic profile, characterized by a general decrease in most ST:STUDY_SUMMARY lipid classes and an increase in glycerophospholipids with ether substituents, ST:STUDY_SUMMARY hexosylceramides, and cholesterol esters compared to the control. These results ST:STUDY_SUMMARY underscore the relevance of the untargeted multiplatform approach to obtain a ST:STUDY_SUMMARY comprehensive overview of the alterations within the renal metabolic map, paving ST:STUDY_SUMMARY the way for further exploration of the molecular mechanisms underlying CKD. ST:INSTITUTE Universidad CEU San Pablo ST:LAST_NAME Rupérez ST:FIRST_NAME Francisco ST:ADDRESS Campus Montepríncipe. Ctra. M-501 km 0 ST:EMAIL ruperez@ceu.es ST:PHONE 913724753 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - 1WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=1WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=1WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=2WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=2WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=3WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=3WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=4WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=4WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=5WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=5WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=6WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=6WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=12WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=12WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 13WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=13WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 13WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=13WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=1WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=1WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=2WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=2WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=3WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=3WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=4WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=4WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=5WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=5WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=6WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=6WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=12WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=12WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 13WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=13WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 13WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=13WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=1WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=1WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=2WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=2WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=3WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=3WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=4WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=4WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=5WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=5WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=6WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=12WT-UUO_GCMS.mzXML #COLLECTION CO:COLLECTION_SUMMARY All animal-related procedures and sample collections were conducted at the CO:COLLECTION_SUMMARY Centro de Biología Molecular "Severo Ochoa" (CBMSO) in accordance with the CO:COLLECTION_SUMMARY guidelines outlined in Directive 2010/63/EU of the European Parliament, known as CO:COLLECTION_SUMMARY the Guide for the Care and Use of Laboratory Animals. The study (PROEX 098.0/22) CO:COLLECTION_SUMMARY received approval from the CBMSO Ethics Committee for Animal Experimentation, CO:COLLECTION_SUMMARY the ethics committee of the Consejo Superior de Investigaciones Científicas CO:COLLECTION_SUMMARY (CSIC) and the Regulatory Unit for Animal Experimental Procedures of the CO:COLLECTION_SUMMARY Comunidad de Madrid. Mice were housed in a specific pathogen-free animal CO:COLLECTION_SUMMARY facility at CBMSO, adhering to EU regulations. CO:SAMPLE_TYPE Kidney #TREATMENT TR:TREATMENT_SUMMARY The UUO murine model (UUO7d) was stablished using the surgical procedure TR:TREATMENT_SUMMARY described in previous studies. Briefly, mice were anesthetized with isoflurane TR:TREATMENT_SUMMARY and the left ureter was doubly ligated with subsequent cutting between the TR:TREATMENT_SUMMARY ligatures. Adequate analgesia was provided to these animals after surgery and TR:TREATMENT_SUMMARY sacrifice was carried out after seven days using CO2 overdose. Control and TR:TREATMENT_SUMMARY obstructed kidneys were harvested following perfusion with PBS. Kidney samples TR:TREATMENT_SUMMARY were categorized into two experimental groups: wild-type control (WTCT) and TR:TREATMENT_SUMMARY wild-type obstruction (WTOBS), each containing 8 samples. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY All analytical-grade organic solvents and chemicals utilized in this study were SP:SAMPLEPREP_SUMMARY obtained from Merck/Sigma-Aldrich (Germany), VWR International/BHD Prolabo SP:SAMPLEPREP_SUMMARY (Spain) and Agilent Technologies (USA). Sialylation-grade pyridine was procured SP:SAMPLEPREP_SUMMARY from VWR International BHD Prolabo (Spain). Reference mass solutions for LC-MS SP:SAMPLEPREP_SUMMARY and CE-MS were obtained from Agilent Technologies. Deionized water (Milli-Q) SP:SAMPLEPREP_SUMMARY utilized throughout the study was obtained using a Milli-Q PLUS system from SP:SAMPLEPREP_SUMMARY Millipore (Austria). Sample treatment Each kidney was dissected both lengthwise SP:SAMPLEPREP_SUMMARY and crosswise to obtain four equal pieces. These sections were promptly frozen SP:SAMPLEPREP_SUMMARY in liquid nitrogen and stored at -80ºC until further analysis. Sample treatment SP:SAMPLEPREP_SUMMARY For tissue disruption and homogenization, a cold methanol solution of 50% was SP:SAMPLEPREP_SUMMARY added to achieve a tissue-weight volume ratio of 1:10. Homogenization was SP:SAMPLEPREP_SUMMARY conducted using a TissueLyser LT bead-mill homogenizer device (QIAGEN, Hilden, SP:SAMPLEPREP_SUMMARY Germany) with 2.8 mm (mean diameter) steel beads 1. The homogenization process SP:SAMPLEPREP_SUMMARY entailed vibrating at a maximum power of 50Hz for 5 minutes, carried out over 4 SP:SAMPLEPREP_SUMMARY cycles, with a 1-minute interval on ice between each cycle. The weight range of SP:SAMPLEPREP_SUMMARY the kidney tissue varied between 25 and 50 mg (WTCT, 32–52.8 mg; WTOBS, SP:SAMPLEPREP_SUMMARY 27.4–50.5 mg). Once the homogenate was obtained, the metabolite extraction SP:SAMPLEPREP_SUMMARY process followed different protocols depending on the analytical platform used SP:SAMPLEPREP_SUMMARY 2,3. For CE-MS analysis, 100 µL of 0.2M formic acid was added to 100 µL of SP:SAMPLEPREP_SUMMARY homogenate, followed by vortex-mixing and centrifugation (16,000×g for 10 SP:SAMPLEPREP_SUMMARY minutes at 4 °C). The resulting supernatant was then transferred to a SP:SAMPLEPREP_SUMMARY Centrifree ultracentrifugation device (Milipore Ireland Ltd., Cork, Ireland) SP:SAMPLEPREP_SUMMARY equipped with a 30-kDa protein cutoff filter for deproteinization through SP:SAMPLEPREP_SUMMARY centrifugation (2000×g for 70 minutes at 4 °C). The filtrate obtained was SP:SAMPLEPREP_SUMMARY transferred to a chromacol vial and subsequently evaporated using a SpeedVac SP:SAMPLEPREP_SUMMARY Concentrator (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the residue SP:SAMPLEPREP_SUMMARY was resuspended in 50 μL of 0.1 M formic acid containing 0.2 mM methionine SP:SAMPLEPREP_SUMMARY sulfone as an internal standard (IS). In the case of LC-MS and GC-MS analysis, SP:SAMPLEPREP_SUMMARY 320 μL of cold methanol was added to 100 μL of homogenate, followed by the SP:SAMPLEPREP_SUMMARY addition of 80 μL of methyl tert-butyl ether. The mixture was vortex-mixed for SP:SAMPLEPREP_SUMMARY 1 hour at room temperature. Subsequently, the samples were centrifuged at 4000 g SP:SAMPLEPREP_SUMMARY at 20ºC for 20 minutes, and the resulting supernatants were divided for LC-MS SP:SAMPLEPREP_SUMMARY and GC-MS analysis. For LC-MS analysis, 100 μL of the supernatant was directly SP:SAMPLEPREP_SUMMARY injected into the UHPLC-MS system. Regarding GC-MS analysis, 300 μL of the SP:SAMPLEPREP_SUMMARY supernatant was transferred to a chromacol vial and evaporated to dryness using SP:SAMPLEPREP_SUMMARY a SpeedVac Concentrator system. The derivatization process commenced by SP:SAMPLEPREP_SUMMARY reconstituting the samples with 20 μL of O-methoxyamine hydrochloride (15 SP:SAMPLEPREP_SUMMARY ng/mL) in pyridine for the methoximation step. After vigorous ultrasonication SP:SAMPLEPREP_SUMMARY and vortex-mixing, the vials were incubated in the dark at room temperature for SP:SAMPLEPREP_SUMMARY 16 hours. Subsequently, for the silylation step, 20 μL of BSTFA:TMCS (99:1) was SP:SAMPLEPREP_SUMMARY added, followed by vortex-mixing for 5 minutes and incubation at 70ºC in the SP:SAMPLEPREP_SUMMARY oven for 1 hour. Finally, 100 μL of heptane containing 20 ppm of tricosane (IS) SP:SAMPLEPREP_SUMMARY was added. It is worth mentioning that due to the limited availability of SP:SAMPLEPREP_SUMMARY samples, the GC-MS analysis was performed with only 6 samples in each group. SP:SAMPLEPREP_SUMMARY Quality control samples (QCs) were prepared for each analytical technique by SP:SAMPLEPREP_SUMMARY pooling equal volumes of each homogenized kidney tissue. Blank samples, which SP:SAMPLEPREP_SUMMARY lacked the analyte of interest but underwent the same extraction protocols, were SP:SAMPLEPREP_SUMMARY also prepared alongside the experimental samples. These blank samples were SP:SAMPLEPREP_SUMMARY analyzed at the beginning and at end of the analytical sequence. QC samples were SP:SAMPLEPREP_SUMMARY injected at the beginning of the sequence to condition and stabilize the SP:SAMPLEPREP_SUMMARY analysis system, and subsequently after every 5-6 experimental samples SP:SAMPLEPREP_SUMMARY throughout the run to assess the stability and performance of the analysis, and SP:SAMPLEPREP_SUMMARY finally, at the end of the analysis sequence. All experimental samples were SP:SAMPLEPREP_SUMMARY randomized prior to sample preparation and analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY To perform LC-MS metabolomics analysis, an Agilent Technologies 1290 Infinity II CH:CHROMATOGRAPHY_SUMMARY UHPLC system was employed, coupled with a 6545-quadrupole time-of-flight (QTOF) CH:CHROMATOGRAPHY_SUMMARY mass spectrometer (Agilent Technologies). The analysis encompassed both ESI+ CH:CHROMATOGRAPHY_SUMMARY (positive) and ESI- (negative) modes, enabling the detection of a wide range of CH:CHROMATOGRAPHY_SUMMARY metabolite ions. The analysis conditions were consistent with those previously CH:CHROMATOGRAPHY_SUMMARY described, 1.5 μL of samples were injected with a multiwash option using the CH:CHROMATOGRAPHY_SUMMARY Agilent 1290 Infinity II Multisampler system with a sample temperature of 15ºC. CH:CHROMATOGRAPHY_SUMMARY Reversed-phase chromatography was used with an InfinityLab Poroshell 120 EC-C8 CH:CHROMATOGRAPHY_SUMMARY (2.1 x 150 mm, 2.7 µm) column and a suitable guard column (Agilent CH:CHROMATOGRAPHY_SUMMARY Technologies) at 60ºC. Mobile phases for the positive ionization mode were CH:CHROMATOGRAPHY_SUMMARY composed by, for aqueous phase (solvent A), 10 mM ammonium formate in Milli-Q CH:CHROMATOGRAPHY_SUMMARY water and for organic phase (solvent B), 10mM ammonium formate in CH:CHROMATOGRAPHY_SUMMARY methanol/isopropanol (85/15, v/v) with a flow rate of 0.5 mL/min. The mobile CH:CHROMATOGRAPHY_SUMMARY phases gradient started at 75% of solvent B, increasing to 96% B at minute 23 CH:CHROMATOGRAPHY_SUMMARY and kept for 8 min. The gradient then increased to 100% of solvent B by minute CH:CHROMATOGRAPHY_SUMMARY 31.5 and was maintained until minute 32.5. At minute 33, the initial condition CH:CHROMATOGRAPHY_SUMMARY was returned, followed by a 7 min-re-equilibration time, with a total run time CH:CHROMATOGRAPHY_SUMMARY of 40 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C8 (150 x 2.1mm,2.7um) CH:SOLVENT_A 100% water; 10 mM ammonium formate CH:SOLVENT_B 85% methanol/15% isopropanol; 10mM ammonium formate CH:FLOW_GRADIENT The mobile phases gradient started at 75% of solvent B, increasing to 96% B at CH:FLOW_GRADIENT minute 23 and kept for 8 min. The gradient then increased to 100% of solvent B CH:FLOW_GRADIENT by minute 31.5 and was maintained until minute 32.5. At minute 33, the initial CH:FLOW_GRADIENT condition was returned, followed by a 7 min-re-equilibration time, with a total CH:FLOW_GRADIENT run time of 40 min. CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 60 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The parameters set in the mass spectrometer for the analysis in the positive and MS:MS_COMMENTS negative mode were as follows: 3500 V capillary voltage, 175 V fragmentor, 65 V MS:MS_COMMENTS skimmer, 750 V octupole radio frequency voltage, 11 L/min for drying gas flow MS:MS_COMMENTS rate at 290 ºC gas temperature, and 40 psi nebulizer pressure, 11L/min sheath MS:MS_COMMENTS gas flow, and 370ºC sheath gas temperature. The MS operated in full scan mode MS:MS_COMMENTS from 100 to 1700 m/z. The reference masses used over the whole analysis were m/z MS:MS_COMMENTS 121.0509 (protonated purine), m/z 149.0233 (protonated phtalic anhydride), and MS:MS_COMMENTS m/z 922.0098 (protonated HP-921) for positive ionization mode. An automated MS:MS_COMMENTS Calibrant Delivery System (CDS) constantly infused these reference masses at a MS:MS_COMMENTS 0.8 mL/min flow rate to allow constant mass correction, using a Dual Agilent Jet MS:MS_COMMENTS Stream Electrospray Ionization (Dual AJS ESI) source for continuously MS:MS_COMMENTS introducing calibrant solution. MS:MS_RESULTS_FILE ST003652_AN005999_Results.txt UNITS:Peak area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END