#METABOLOMICS WORKBENCH Davidobe12_20200327_124023 DATATRACK_ID:1961 STUDY_ID:ST001369 ANALYSIS_ID:AN002283 PROJECT_ID:000000 VERSION 1 CREATED_ON April 29, 2020, 10:46 am #PROJECT PR:PROJECT_TITLE Metabolomics analysis of serum samples from patients during 2 years of IAT PR:PROJECT_TITLE trial, double blind placebo controled study PR:PROJECT_TYPE Study the changes produced by the use of AIT treatment or placebo PR:PROJECT_SUMMARY 47 patients were enrolled in a double-blind, placebo-controlled, multicenter PR:PROJECT_SUMMARY trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). PR:PROJECT_SUMMARY Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 PR:PROJECT_SUMMARY patients who finished the trial. Additionally, serum and PBMCs samples from PR:PROJECT_SUMMARY these samples were analyzed by metabolomics and transcriptomics, respectively. PR:PROJECT_SUMMARY Based on their sensitization level, 22 patients were grouped in Mono and Poli PR:PROJECT_SUMMARY groups, excluding epithelial allergic patients. Individuals were studied based PR:PROJECT_SUMMARY on their treatment in Active and Placebo and their sensitization level. PR:INSTITUTE Institute of Applied Molecular Medicine, CEU; The Centre of Metabolomics and PR:INSTITUTE Bioanalysis, CEU PR:LAST_NAME Barber Hernández PR:FIRST_NAME Domingo PR:ADDRESS Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España PR:EMAIL domingo.barberhernandez@ceu.es PR:PHONE Tlf: 91 372 47 00 ext. 4662 #STUDY ST:STUDY_TITLE Grass pollen sublingual immunotherapy treatment induces transcriptomic and ST:STUDY_TITLE metabolic changes due to AIT treatment ST:STUDY_SUMMARY 47 patients were enrolled in a double-blind, placebo-controlled, multicenter ST:STUDY_SUMMARY trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). ST:STUDY_SUMMARY Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 ST:STUDY_SUMMARY patients who finished the trial. Additionally, serum and PBMCs samples from ST:STUDY_SUMMARY these samples were analyzed by metabolomics and transcriptomics, respectively. ST:STUDY_SUMMARY Based on their sensitization level, 22 patients were grouped in Mono and Poli ST:STUDY_SUMMARY groups, excluding epithelial allergic patients. Individuals were studied based ST:STUDY_SUMMARY on their treatment in Active and Placebo and their sensitization level. For ST:STUDY_SUMMARY metabolomics, samples were analyzed by Liquid and Gas Chromatography coupled to ST:STUDY_SUMMARY Mass Spectrometry (LC-MS and GC-MS, respectively). ST:INSTITUTE Institute of Applied Molecular Medicine, CEU; The Centre of Metabolomics and ST:INSTITUTE Bioanalysis, CEU ST:LAST_NAME Obeso Montero ST:FIRST_NAME David ST:ADDRESS Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España ST:EMAIL david.obesomontero@beca.ceu.es ST:PHONE Tlf: 91 372 47 00 ext. 4662 ST:NUM_GROUPS 2 main groups: Active and Placebo, and 2 subgroups: Monosensitized and ST:NUM_GROUPS Polisensitized patients. ST:TOTAL_SUBJECTS 22 ST:STUDY_COMMENTS https://www.ceu.es;http://www.metabolomica.uspceu.es #SUBJECT SU:SUBJECT_TYPE Human 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 Subject 1 P1_t0 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P1_t0.d SUBJECT_SAMPLE_FACTORS Subject 2 P3_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P3_t0.d SUBJECT_SAMPLE_FACTORS Subject 3 P4_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P4_t0.d SUBJECT_SAMPLE_FACTORS Subject 4 P8_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P8_t0.d SUBJECT_SAMPLE_FACTORS Subject 5 P9_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P9_t0.d SUBJECT_SAMPLE_FACTORS Subject 6 P11_t0 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P11_t0.d SUBJECT_SAMPLE_FACTORS Subject 7 P12_t0 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P12_t0.d SUBJECT_SAMPLE_FACTORS Subject 8 P13_t0 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P13_t0.d SUBJECT_SAMPLE_FACTORS Subject 9 P14_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P14_t0.d SUBJECT_SAMPLE_FACTORS Subject 10 P15_t0 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P15_t0.d SUBJECT_SAMPLE_FACTORS Subject 11 P16_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P16_t0.d SUBJECT_SAMPLE_FACTORS Subject 12 P17_t0 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P17_t0.d SUBJECT_SAMPLE_FACTORS Subject 13 P20_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P20_t0.d SUBJECT_SAMPLE_FACTORS Subject 14 P21_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P21_t0.d SUBJECT_SAMPLE_FACTORS Subject 15 P22_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P22_t0.d SUBJECT_SAMPLE_FACTORS Subject 16 P23_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P23_t0.d SUBJECT_SAMPLE_FACTORS Subject 17 P24_t0 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P24_t0.d SUBJECT_SAMPLE_FACTORS Subject 18 P25_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P25_t0.d SUBJECT_SAMPLE_FACTORS Subject 19 P26_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P26_t0.d SUBJECT_SAMPLE_FACTORS Subject 20 P27_t0 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P27_t0.d SUBJECT_SAMPLE_FACTORS Subject 21 P28_t0 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P28_t0.d SUBJECT_SAMPLE_FACTORS Subject 22 P30_t0 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P30_t0.d SUBJECT_SAMPLE_FACTORS Subject 1 P1_t2 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P1_t2.d SUBJECT_SAMPLE_FACTORS Subject 2 P3_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P3_t2.d SUBJECT_SAMPLE_FACTORS Subject 3 P4_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P4_t2.d SUBJECT_SAMPLE_FACTORS Subject 4 P8_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P8_t2.d SUBJECT_SAMPLE_FACTORS Subject 5 P9_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P9_t2.d SUBJECT_SAMPLE_FACTORS Subject 6 P11_t2 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P11_t2.d SUBJECT_SAMPLE_FACTORS Subject 7 P12_t2 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P12_t2.d SUBJECT_SAMPLE_FACTORS Subject 8 P13_t2 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P13_t2.d SUBJECT_SAMPLE_FACTORS Subject 9 P14_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P14_t2.d SUBJECT_SAMPLE_FACTORS Subject 10 P15_t2 Treatment:Active | Sensitization:Poli RAW_FILE_NAME=P15_t2.d SUBJECT_SAMPLE_FACTORS Subject 11 P16_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P16_t2.d SUBJECT_SAMPLE_FACTORS Subject 12 P17_t2 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P17_t2.d SUBJECT_SAMPLE_FACTORS Subject 13 P20_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P20_t2.d SUBJECT_SAMPLE_FACTORS Subject 14 P21_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P21_t2.d SUBJECT_SAMPLE_FACTORS Subject 15 P22_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P22_t2.d SUBJECT_SAMPLE_FACTORS Subject 16 P23_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P23_t2.d SUBJECT_SAMPLE_FACTORS Subject 17 P24_t2 Treatment:Placebo | Sensitization:Poli RAW_FILE_NAME=P24_t2.d SUBJECT_SAMPLE_FACTORS Subject 18 P25_T2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P25_t2.d SUBJECT_SAMPLE_FACTORS Subject 19 P26_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P26_t2.d SUBJECT_SAMPLE_FACTORS Subject 20 P27_T2 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P27_t2.d SUBJECT_SAMPLE_FACTORS Subject 21 P28_t2 Treatment:Active | Sensitization:Mono RAW_FILE_NAME=P28_t2.d SUBJECT_SAMPLE_FACTORS Subject 22 P30_t2 Treatment:Placebo | Sensitization:Mono RAW_FILE_NAME=P30_t2.d #COLLECTION CO:COLLECTION_SUMMARY For immunological analyses and metabolomics, serum samples were obtained and CO:COLLECTION_SUMMARY stored at -20°C until analysis. In the case of transcriptomics, Peripheral CO:COLLECTION_SUMMARY blood mononuclear cells (PBMCs) were isolated from whole blood and stored at CO:COLLECTION_SUMMARY -20°C in Buffer RLT until analysis. CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -20℃ #TREATMENT TR:TREATMENT_SUMMARY Subjects were randomized (1:1) during autumn 2013 to receive either active TR:TREATMENT_SUMMARY treatment with GRAZAX® (Phleum pratense, 75,000SQ‐T tablets ALK, Hørsholm, TR:TREATMENT_SUMMARY Denmark) or placebo as Investigational Medical Products (IMP) during 2 years. TR:TREATMENT_SUMMARY Twenty-five patients were assigned to daily sublingual administration of TR:TREATMENT_SUMMARY GRAZAX®, while the rest received placebo tablets that were similar to the TR:TREATMENT_SUMMARY active IMP with regard to appearance, smell and taste. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For LC-MS, proteins were removed adding 300 µL of cold (-20 °C) SP:SAMPLEPREP_SUMMARY methanol:ethanol (1:1) to 100 µL of sample. Samples were then vortex-mixed and SP:SAMPLEPREP_SUMMARY stored on ice for 5 min. Supernatant containing the metabolites was separated SP:SAMPLEPREP_SUMMARY from the pellet by centrifugation (16,000× g for 20 min at 4 °C), and put into SP:SAMPLEPREP_SUMMARY a LC vial for analysis. For GC-MS, serum samples were first deproteinized using SP:SAMPLEPREP_SUMMARY cold acetonitrile (ACN) in a 3:1 proportion (120 μL of ACN were added to 40 μL SP:SAMPLEPREP_SUMMARY of serum). Samples were kept on ice for 5 minutes. Afterwards, the metabolites SP:SAMPLEPREP_SUMMARY were separated by centrifugation (10 min at 15,400 x g and 4°C). 100 µL of the SP:SAMPLEPREP_SUMMARY resulting supernatant were transferred to a GC vial with insert and were SP:SAMPLEPREP_SUMMARY evaporated to dryness for 2 hours at 30°C (SpeedVac Concentrator, Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific, Waltham, MA, USA). Afterwards, a methoximation was performed by SP:SAMPLEPREP_SUMMARY addition of 10 µL of O-methoxyamine hydrochloride 15mg/mL in pyridine, with the SP:SAMPLEPREP_SUMMARY purpose of protecting the reactive oxygen groups in the metabolites. The mixture SP:SAMPLEPREP_SUMMARY was vigorously vortex-mixed (1 minute each vial), ultrasonicated (3 times, 20 SP:SAMPLEPREP_SUMMARY seconds each) and further vortexed another 2 minutes. Then, the samples were SP:SAMPLEPREP_SUMMARY left in darkness at room temperature for 16 h for the completion of the SP:SAMPLEPREP_SUMMARY methoximation reaction. Finally, the samples were derivatized by the addition of SP:SAMPLEPREP_SUMMARY 10 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% SP:SAMPLEPREP_SUMMARY trimethylchlorosilane (TMCS). SP:PROCESSING_METHOD Protein precipitation and metabolite extraction SP:PROCESSING_STORAGE_CONDITIONS On ice #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For LC-MS, 10 μL of sample were injected into a Discovery HS C18 column (2.1 mm CH:CHROMATOGRAPHY_SUMMARY × 150 mm, 3.0 μm; Supelco, Sigma Aldrich, Germany), with a guard column CH:CHROMATOGRAPHY_SUMMARY Discovery® HS C18 (2 cm × 2.1 mm, 3 μm; Supelco, Sigma Aldrich, Germany), CH:CHROMATOGRAPHY_SUMMARY both maintained at 40 °C. The flow rate was set at 0.6 mL/min. The gradient CH:CHROMATOGRAPHY_SUMMARY elution involved a mobile phase consisting of (A) 0.1% formic acid (FA) in water CH:CHROMATOGRAPHY_SUMMARY and (B) 0.1% FA in acetonitrile (ACN). The initial condition was set at 25% B, CH:CHROMATOGRAPHY_SUMMARY increasing to 95% B in 35 min, followed by re-equilibration for 1 min, finally CH:CHROMATOGRAPHY_SUMMARY it was held for 9 min in initial conditions. Flow rate was set at 0.6 mL/min, CH:CHROMATOGRAPHY_SUMMARY and 10 μL of samples were injected. The electrospray source ionization (ESI) CH:CHROMATOGRAPHY_SUMMARY data were acquired in positive and negative ion mode, respectively. The CH:CHROMATOGRAPHY_SUMMARY capillary voltage was set at 3,500 for ESI (+) and 4,000V for ESI (−). The CH:CHROMATOGRAPHY_SUMMARY drying gas flow rate was 10.5 L/min at 330 °C and gas nebulizer at 52 psi; CH:CHROMATOGRAPHY_SUMMARY fragmentor voltage was 175 V; skimmer and octopole radio frequency voltage (OCT CH:CHROMATOGRAPHY_SUMMARY RF Vpp) were set to 65 and 750 V. Data were collected in the centroid mode at a CH:CHROMATOGRAPHY_SUMMARY scan rate of 1.2 spectra per second. Mass spectrometry detection was performed CH:CHROMATOGRAPHY_SUMMARY in full scan from 100 to 1200 m/z for both, positive and negative ESI mode. The CH:CHROMATOGRAPHY_SUMMARY reference m/z ions were purine (121.0508) and HP-0921 (922.0097) for ESI (+), CH:CHROMATOGRAPHY_SUMMARY whereas TFA NH4 (119.0363) and HP-0921 (966.0007) for ESI (−). These masses CH:CHROMATOGRAPHY_SUMMARY were continuously infused into the system to allow constant mass correction. CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed in separate runs. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1200 CH:COLUMN_NAME Discovery HS C18 column (2.1 mm × 150 mm, 3.0 µm; Supelco, Sigma Aldrich, Germany), with a guard column Discovery® HS C18 (2 cm × 2.1 mm, 3 µm; Supelco, Sigma Aldrich, Germany), CH:COLUMN_NAME Germany), with a guard column Discovery® HS C18 (2 cm × 2.1 mm, 3 μm; CH:COLUMN_NAME Supelco, Sigma Aldrich, Germany), CH:FLOW_GRADIENT The initial condition was set at 25% B, increasing to 95% B in 35 min, followed CH:FLOW_GRADIENT by re-equilibration for 1 min, finally it was held for 9 min in initial CH:FLOW_GRADIENT conditions CH:FLOW_RATE 0.6ml/min CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A 0.1% formic acid (FA) in water CH:SOLVENT_B 0.1% FA in acetonitrile (ACN) CH:INJECTION_TEMPERATURE 4 CH:SAMPLE_INJECTION 10 μL CH:ANALYTICAL_TIME 45 min CH:CAPILLARY_VOLTAGE 3500 for ESI (+) #ANALYSIS AN:ANALYSIS_TYPE MS AN:DATA_FORMAT .d #MS MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS LC−MS analysis was performed on an Agilent HPLC system (1200 series, Agilent MS:MS_COMMENTS Technologies, Waldbronn, Germany), equipped with a degasser, two binary pumps, MS:MS_COMMENTS and a thermostated autosampler coupled with quadrupole-time of flight analyzer MS:MS_COMMENTS (Q-TOF), LC-MS (6520) system (Agilent Technologies, Waldbronn, Germany) MS:CAPILLARY_VOLTAGE 3,500 for ESI (+) MS:DRY_GAS_FLOW 10.5 L/min MS:DRY_GAS_TEMP 330 °C MS:FRAGMENT_VOLTAGE 175 V MS:NEBULIZER 52 psi MS:OCTPOLE_VOLTAGE 750V MS:MS_RESULTS_FILE ST001369_AN002283_Results.txt UNITS:peak area Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END