{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001369","ANALYSIS_ID":"AN002283","VERSION":"1","CREATED_ON":"April 29, 2020, 10:46 am"},

"PROJECT":{"PROJECT_TITLE":"Metabolomics analysis of serum samples from patients during 2 years of IAT trial, double blind placebo controled study","PROJECT_TYPE":"Study the changes produced by the use of AIT treatment or placebo","PROJECT_SUMMARY":"47 patients were enrolled in a double-blind, placebo-controlled, multicenter trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 patients who finished the trial. Additionally, serum and PBMCs samples from these samples were analyzed by metabolomics and transcriptomics, respectively. Based on their sensitization level, 22 patients were grouped in Mono and Poli groups, excluding epithelial allergic patients. Individuals were studied based on their treatment in Active and Placebo and their sensitization level.","INSTITUTE":"Institute of Applied Molecular Medicine, CEU; The Centre of Metabolomics and Bioanalysis, CEU","LAST_NAME":"Barber Hernández","FIRST_NAME":"Domingo","ADDRESS":"Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España","EMAIL":"domingo.barberhernandez@ceu.es","PHONE":"Tlf: 91 372 47 00 ext. 4662"},

"STUDY":{"STUDY_TITLE":"Grass pollen sublingual immunotherapy treatment induces transcriptomic and metabolic changes due to AIT treatment","STUDY_SUMMARY":"47 patients were enrolled in a double-blind, placebo-controlled, multicenter trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 patients who finished the trial. Additionally, serum and PBMCs samples from these samples were analyzed by metabolomics and transcriptomics, respectively. Based on their sensitization level, 22 patients were grouped in Mono and Poli groups, excluding epithelial allergic patients. Individuals were studied based on their treatment in Active and Placebo and their sensitization level. For metabolomics, samples were analyzed by Liquid and Gas Chromatography coupled to Mass Spectrometry (LC-MS and GC-MS, respectively).","INSTITUTE":"Institute of Applied Molecular Medicine, CEU; The Centre of Metabolomics and Bioanalysis, CEU","LAST_NAME":"Obeso Montero","FIRST_NAME":"David","ADDRESS":"Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España","EMAIL":"david.obesomontero@beca.ceu.es","PHONE":"Tlf: 91 372 47 00 ext. 4662","NUM_GROUPS":"2 main groups: Active and Placebo, and 2 subgroups: Monosensitized and Polisensitized patients.","TOTAL_SUBJECTS":"22","STUDY_COMMENTS":"https://www.ceu.es;http://www.metabolomica.uspceu.es"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Subject 1",
"Sample ID":"P1_t0",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P1_t0.d"}
},
{
"Subject ID":"Subject 2",
"Sample ID":"P3_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P3_t0.d"}
},
{
"Subject ID":"Subject 3",
"Sample ID":"P4_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P4_t0.d"}
},
{
"Subject ID":"Subject 4",
"Sample ID":"P8_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P8_t0.d"}
},
{
"Subject ID":"Subject 5",
"Sample ID":"P9_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P9_t0.d"}
},
{
"Subject ID":"Subject 6",
"Sample ID":"P11_t0",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P11_t0.d"}
},
{
"Subject ID":"Subject 7",
"Sample ID":"P12_t0",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P12_t0.d"}
},
{
"Subject ID":"Subject 8",
"Sample ID":"P13_t0",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P13_t0.d"}
},
{
"Subject ID":"Subject 9",
"Sample ID":"P14_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P14_t0.d"}
},
{
"Subject ID":"Subject 10",
"Sample ID":"P15_t0",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P15_t0.d"}
},
{
"Subject ID":"Subject 11",
"Sample ID":"P16_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P16_t0.d"}
},
{
"Subject ID":"Subject 12",
"Sample ID":"P17_t0",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P17_t0.d"}
},
{
"Subject ID":"Subject 13",
"Sample ID":"P20_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P20_t0.d"}
},
{
"Subject ID":"Subject 14",
"Sample ID":"P21_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P21_t0.d"}
},
{
"Subject ID":"Subject 15",
"Sample ID":"P22_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P22_t0.d"}
},
{
"Subject ID":"Subject 16",
"Sample ID":"P23_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P23_t0.d"}
},
{
"Subject ID":"Subject 17",
"Sample ID":"P24_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P24_t0.d"}
},
{
"Subject ID":"Subject 18",
"Sample ID":"P25_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P25_t0.d"}
},
{
"Subject ID":"Subject 19",
"Sample ID":"P26_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P26_t0.d"}
},
{
"Subject ID":"Subject 20",
"Sample ID":"P27_t0",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P27_t0.d"}
},
{
"Subject ID":"Subject 21",
"Sample ID":"P28_t0",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P28_t0.d"}
},
{
"Subject ID":"Subject 22",
"Sample ID":"P30_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P30_t0.d"}
},
{
"Subject ID":"Subject 1",
"Sample ID":"P1_t2",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P1_t2.d"}
},
{
"Subject ID":"Subject 2",
"Sample ID":"P3_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P3_t2.d"}
},
{
"Subject ID":"Subject 3",
"Sample ID":"P4_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P4_t2.d"}
},
{
"Subject ID":"Subject 4",
"Sample ID":"P8_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P8_t2.d"}
},
{
"Subject ID":"Subject 5",
"Sample ID":"P9_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P9_t2.d"}
},
{
"Subject ID":"Subject 6",
"Sample ID":"P11_t2",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P11_t2.d"}
},
{
"Subject ID":"Subject 7",
"Sample ID":"P12_t2",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P12_t2.d"}
},
{
"Subject ID":"Subject 8",
"Sample ID":"P13_t2",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P13_t2.d"}
},
{
"Subject ID":"Subject 9",
"Sample ID":"P14_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P14_t2.d"}
},
{
"Subject ID":"Subject 10",
"Sample ID":"P15_t2",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P15_t2.d"}
},
{
"Subject ID":"Subject 11",
"Sample ID":"P16_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P16_t2.d"}
},
{
"Subject ID":"Subject 12",
"Sample ID":"P17_t2",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P17_t2.d"}
},
{
"Subject ID":"Subject 13",
"Sample ID":"P20_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P20_t2.d"}
},
{
"Subject ID":"Subject 14",
"Sample ID":"P21_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P21_t2.d"}
},
{
"Subject ID":"Subject 15",
"Sample ID":"P22_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P22_t2.d"}
},
{
"Subject ID":"Subject 16",
"Sample ID":"P23_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P23_t2.d"}
},
{
"Subject ID":"Subject 17",
"Sample ID":"P24_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P24_t2.d"}
},
{
"Subject ID":"Subject 18",
"Sample ID":"P25_T2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P25_t2.d"}
},
{
"Subject ID":"Subject 19",
"Sample ID":"P26_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P26_t2.d"}
},
{
"Subject ID":"Subject 20",
"Sample ID":"P27_T2",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P27_t2.d"}
},
{
"Subject ID":"Subject 21",
"Sample ID":"P28_t2",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P28_t2.d"}
},
{
"Subject ID":"Subject 22",
"Sample ID":"P30_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P30_t2.d"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For immunological analyses and metabolomics, serum samples were obtained and stored at -20°C until analysis. In the case of transcriptomics, Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and stored at -20°C in Buffer RLT until analysis.","SAMPLE_TYPE":"Blood (serum)","STORAGE_CONDITIONS":"-20℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Subjects were randomized (1:1) during autumn 2013 to receive either active treatment with GRAZAX® (Phleum pratense, 75,000SQ‐T tablets ALK, Hørsholm, Denmark) or placebo as Investigational Medical Products (IMP) during 2 years. Twenty-five patients were assigned to daily sublingual administration of GRAZAX®, while the rest received placebo tablets that were similar to the active IMP with regard to appearance, smell and taste."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"For LC-MS, proteins were removed adding 300 µL of cold (-20 °C) methanol:ethanol (1:1) to 100 µL of sample. Samples were then vortex-mixed and stored on ice for 5 min. Supernatant containing the metabolites was separated from the pellet by centrifugation (16,000× g for 20 min at 4 °C), and put into a LC vial for analysis. For GC-MS, serum samples were first deproteinized using cold acetonitrile (ACN) in a 3:1 proportion (120 μL of ACN were added to 40 μL of serum). Samples were kept on ice for 5 minutes. Afterwards, the metabolites were separated by centrifugation (10 min at 15,400 x g and 4°C). 100 µL of the resulting supernatant were transferred to a GC vial with insert and were evaporated to dryness for 2 hours at 30°C (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, a methoximation was performed by addition of 10 µL of O-methoxyamine hydrochloride 15mg/mL in pyridine, with the purpose of protecting the reactive oxygen groups in the metabolites. The mixture was vigorously vortex-mixed (1 minute each vial), ultrasonicated (3 times, 20 seconds each) and further vortexed another 2 minutes. Then, the samples were left in darkness at room temperature for 16 h for the completion of the methoximation reaction. Finally, the samples were derivatized by the addition of 10 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS).","PROCESSING_METHOD":"Protein precipitation and metabolite extraction","PROCESSING_STORAGE_CONDITIONS":"On ice"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"For LC-MS, 10 μL of sample were injected into a 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), both maintained at 40 °C. The flow rate was set at 0.6 mL/min. The gradient elution involved a mobile phase consisting of (A) 0.1% formic acid (FA) in water and (B) 0.1% FA in acetonitrile (ACN). The initial condition was set at 25% B, increasing to 95% B in 35 min, followed by re-equilibration for 1 min, finally it was held for 9 min in initial conditions. Flow rate was set at 0.6 mL/min, and 10 μL of samples were injected. The electrospray source ionization (ESI) data were acquired in positive and negative ion mode, respectively. The capillary voltage was set at 3,500 for ESI (+) and 4,000V for ESI (−). The drying gas flow rate was 10.5 L/min at 330 °C and gas nebulizer at 52 psi; fragmentor voltage was 175 V; skimmer and octopole radio frequency voltage (OCT RF Vpp) were set to 65 and 750 V. Data were collected in the centroid mode at a scan rate of 1.2 spectra per second. Mass spectrometry detection was performed in full scan from 100 to 1200 m/z for both, positive and negative ESI mode. The reference m/z ions were purine (121.0508) and HP-0921 (922.0097) for ESI (+), whereas TFA NH4 (119.0363) and HP-0921 (966.0007) for ESI (−). These masses were continuously infused into the system to allow constant mass correction. Samples were analyzed in separate runs.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Agilent 1200","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), Germany), with a guard column Discovery® HS C18 (2 cm × 2.1 mm, 3 μm; Supelco, Sigma Aldrich, Germany),","FLOW_GRADIENT":"The initial condition was set at 25% B, increasing to 95% B in 35 min, followed by re-equilibration for 1 min, finally it was held for 9 min in initial conditions","FLOW_RATE":"0.6ml/min","COLUMN_TEMPERATURE":"40","SOLVENT_A":"0.1% formic acid (FA) in water","SOLVENT_B":"0.1% FA in acetonitrile (ACN)","INJECTION_TEMPERATURE":"4","SAMPLE_INJECTION":"10 μL","ANALYTICAL_TIME":"45 min","CAPILLARY_VOLTAGE":"3500 for ESI (+)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","DATA_FORMAT":".d"},

"MS":{"INSTRUMENT_NAME":"Agilent 6520 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"LC−MS analysis was performed on an Agilent HPLC system (1200 series, Agilent Technologies, Waldbronn, Germany), equipped with a degasser, two binary pumps, and a thermostated autosampler coupled with quadrupole-time of flight analyzer (Q-TOF), LC-MS (6520) system (Agilent Technologies, Waldbronn, Germany)","CAPILLARY_VOLTAGE":"3,500 for ESI (+)","DRY_GAS_FLOW":"10.5 L/min","DRY_GAS_TEMP":"330 °C","FRAGMENT_VOLTAGE":"175 V","NEBULIZER":"52 psi","OCTPOLE_VOLTAGE":"750V","MS_RESULTS_FILE":"ST001369_AN002283_Results.txt UNITS:peak area Has m/z:Neutral masses Has RT:Yes RT units:Minutes"}

}