{ "METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000980","ANALYSIS_ID":"AN001604","VERSION":"1","CREATED_ON":"June 19, 2018, 12:17 pm"}, "PROJECT":{"PROJECT_TITLE":"Metabolomics analysis of plasma samples from non-allergic subjects and patients with different severity of food allergy","PROJECT_SUMMARY":"Metabolomic analysis of plasma samples from grass pollen allergic patients with different levels of allergic severity to profilin.","INSTITUTE":"Institute of Applied Molecular Medicine and The Centre of Metabolomics and Bioanalysis","DEPARTMENT":"Analytical chemistry","LAST_NAME":"Barber Hernández","FIRST_NAME":"Domingo","ADDRESS":"Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España","EMAIL":"domingo.barberhernandez@ceu.es","PHONE":"+34 91 372 47 00 ext.4662"}, "STUDY":{"STUDY_TITLE":"Metabolomic Analysis of plasma samples from Non-Allergic Subjects and Profilin-Allergic Patients overexposed to Grass Pollen","STUDY_TYPE":"Allergy severity comparison","STUDY_SUMMARY":"Prevalence and severity of allergic diseases have increased worldwide. To date, respiratory allergy phenotypes are not fully characterized and, along with inflammation progression, treatment is increasingly complex and expensive. Profilin sensitization constitutes a good model to study the progression of allergic inflammation. Our aim was to identify the underlying mechanisms and the associated biomarkers of this progression, focusing on severe phenotypes, using transcriptomics and metabolomics. Methods: 25 subjects were included in the study. Plasma samples were analyzed using Gas and Liquid Chromatography coupled to Mass Spectrometry (GC-MS and LC-MS, respectively). Individuals were classified in 4 groups – “non-allergic”, “mild”, “moderate” and “severe” – based on their clinical history, their response to an oral challenge test with profilin, and after a refinement using a mathematical metabolomic model. PBMCs were used for microarray analysis.","INSTITUTE":"The Centre of Metabolomics and Bioanalysis","DEPARTMENT":"Analytical chemistry","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":"913724769","NUM_GROUPS":"4 groups","TOTAL_SUBJECTS":"25 plasma samples"}, "SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"}, "SUBJECT_SAMPLE_FACTORS":[ { "Subject ID":"P1", "Sample ID":"02_005", "Factors":{"Classification":"Non-allergic"} }, { "Subject ID":"P2", "Sample ID":"02_006", "Factors":{"Classification":"Non-allergic"} }, { "Subject ID":"P3", "Sample ID":"02_007", "Factors":{"Classification":"Non-allergic"} }, { "Subject ID":"P4", "Sample ID":"02_008", "Factors":{"Classification":"Non-allergic"} }, { "Subject ID":"P5", "Sample ID":"04_003", "Factors":{"Classification":"Non-allergic"} }, { "Subject ID":"P6", "Sample ID":"04_006", "Factors":{"Classification":"Non-allergic"} }, { "Subject ID":"P7", "Sample ID":"01_001", "Factors":{"Classification":"Mild"} }, { "Subject ID":"P8", "Sample ID":"01_002", "Factors":{"Classification":"Mild"} }, { "Subject ID":"P9", "Sample ID":"02_002", "Factors":{"Classification":"Mild"} }, { "Subject ID":"P10", "Sample ID":"02_003", "Factors":{"Classification":"Mild"} }, { "Subject ID":"P11", "Sample ID":"04_001", "Factors":{"Classification":"Mild"} }, { "Subject ID":"P12", "Sample ID":"01_005", "Factors":{"Classification":"Moderate"} }, { "Subject ID":"P13", "Sample ID":"02_001", "Factors":{"Classification":"Moderate"} }, { "Subject ID":"P14", "Sample ID":"02_004", "Factors":{"Classification":"Moderate"} }, { "Subject ID":"P15", "Sample ID":"03_004", "Factors":{"Classification":"Moderate"} }, { "Subject ID":"P16", "Sample ID":"03_098", "Factors":{"Classification":"Moderate"} }, { "Subject ID":"P17", "Sample ID":"04_002", "Factors":{"Classification":"Moderate"} }, { "Subject ID":"P18", "Sample ID":"03_002", "Factors":{"Classification":"Severe"} }, { "Subject ID":"P19", "Sample ID":"03_003", "Factors":{"Classification":"Severe"} }, { "Subject ID":"P20", "Sample ID":"03_092", "Factors":{"Classification":"Severe"} }, { "Subject ID":"P21", "Sample ID":"03_093", "Factors":{"Classification":"Severe"} }, { "Subject ID":"P22", "Sample ID":"03_094", "Factors":{"Classification":"Severe"} }, { "Subject ID":"P23", "Sample ID":"03_095", "Factors":{"Classification":"Severe"} }, { "Subject ID":"P24", "Sample ID":"03_096", "Factors":{"Classification":"Severe"} }, { "Subject ID":"P25", "Sample ID":"03_099", "Factors":{"Classification":"Severe"} } ], "COLLECTION":{"COLLECTION_SUMMARY":"Whole blood was drawn before the profilin challenge and was divided into two tubes: one for plasma extraction and other for PBMCs isolation. Briefly, 20 ml of peripheral blood were collected to obtain plasma by centrifugation(1500rpm x 5 min.) and PBMCs using Ficoll-Paque (GE Healthcare™) density gradient centrifugation.","SAMPLE_TYPE":"Blood (whole)","COLLECTION_LOCATION":"Hospital Universitario Clínico San Carlos, Madrid, España. Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IP), Madrid, España. Hospital Virgen del Puerto, Plasencia, Cáceres, España. Hospital Universitario HM Sanchinarro, Madrid, España.","VOLUMEORAMOUNT_COLLECTED":"14-16 mL Blood","STORAGE_CONDITIONS":"-80℃","COLLECTION_VIALS":"BD Vacutainer® Heparin Blood Collection Tubes (Ref. BD367871)","STORAGE_VIALS":"Eppendorf 1.5 mL","COLLECTION_TUBE_TEMP":"Room Temperature"}, "TREATMENT":{"TREATMENT_SUMMARY":"No treatment was used in this study."}, "SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"For LC-MS, plasma protein was 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 protein precipitation, 120 μL of cold acetonitrile (ACN) were added to 40 μL of each plasma sample in an Eppendorf and were stored on ice for 5 minutes. Then, metabolites were separated by centrifugation (16,000× g, 15 min, 4 °C). From the resulting supernatant, 100 μL were transferred to GC vial with insert and were evaporated to dryness (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Then, 10 μL of O-methoxyamine hydrochloride in pyridine (15 mg/mL) was added to each GC vial, and the mixture was vigorously vortex-mixed and ultrasonicated. Methoxymation was carried out in darkness, at room temperature for 16 h. Afterwards, 10 μL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) were added as catalyst and the solution was further mixed using the vortex. For silylation process, samples were heated in an oven for 1h at 70 °C. Finally, 100 μL of heptane containing 10 ppm of C18:0 methyl ester (IS) were added to each GC vial and vortex-mixed before GC analysis."}, "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":"Normal phase","INSTRUMENT_NAME":"Agilent 1200","COLUMN_NAME":"Unspecified"}, "ANALYSIS":{"ANALYSIS_TYPE":"MS"}, "MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Agilent 6520 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","CAPILLARY_VOLTAGE":"3500 V","DRY_GAS_FLOW":"10.5 L/min","DRY_GAS_TEMP":"330 °C","FRAGMENT_VOLTAGE":"175 V","MS_RESULTS_FILE":"ST000980_AN001604_Results.txt UNITS:peak area"} }