#METABOLOMICS WORKBENCH Davidobe12_20180613_060330 DATATRACK_ID:1413 STUDY_ID:ST000980 ANALYSIS_ID:AN001604 PROJECT_ID:PR000670 VERSION 1 CREATED_ON June 19, 2018, 12:17 pm #PROJECT PR:PROJECT_TITLE Metabolomics analysis of plasma samples from non-allergic subjects and patients PR:PROJECT_TITLE with different severity of food allergy PR:PROJECT_SUMMARY Metabolomic analysis of plasma samples from grass pollen allergic patients with PR:PROJECT_SUMMARY different levels of allergic severity to profilin. PR:INSTITUTE Institute of Applied Molecular Medicine and The Centre of Metabolomics and PR:INSTITUTE Bioanalysis PR:DEPARTMENT Analytical chemistry 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 +34 91 372 47 00 ext.4662 #STUDY ST:STUDY_TITLE Metabolomic Analysis of plasma samples from Non-Allergic Subjects and ST:STUDY_TITLE Profilin-Allergic Patients overexposed to Grass Pollen ST:STUDY_TYPE Allergy severity comparison ST:STUDY_SUMMARY Prevalence and severity of allergic diseases have increased worldwide. To date, ST:STUDY_SUMMARY respiratory allergy phenotypes are not fully characterized and, along with ST:STUDY_SUMMARY inflammation progression, treatment is increasingly complex and expensive. ST:STUDY_SUMMARY Profilin sensitization constitutes a good model to study the progression of ST:STUDY_SUMMARY allergic inflammation. Our aim was to identify the underlying mechanisms and the ST:STUDY_SUMMARY associated biomarkers of this progression, focusing on severe phenotypes, using ST:STUDY_SUMMARY transcriptomics and metabolomics. Methods: 25 subjects were included in the ST:STUDY_SUMMARY study. Plasma samples were analyzed using Gas and Liquid Chromatography coupled ST:STUDY_SUMMARY to Mass Spectrometry (GC-MS and LC-MS, respectively). Individuals were ST:STUDY_SUMMARY classified in 4 groups – “non-allergic”, “mild”, “moderate” and ST:STUDY_SUMMARY “severe” – based on their clinical history, their response to an oral ST:STUDY_SUMMARY challenge test with profilin, and after a refinement using a mathematical ST:STUDY_SUMMARY metabolomic model. PBMCs were used for microarray analysis. ST:INSTITUTE The Centre of Metabolomics and Bioanalysis ST:DEPARTMENT Analytical chemistry 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 913724769 ST:NUM_GROUPS 4 groups ST:TOTAL_SUBJECTS 25 plasma samples #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]Additional sample data SUBJECT_SAMPLE_FACTORS P1 02_005 Classification:Non-allergic SUBJECT_SAMPLE_FACTORS P2 02_006 Classification:Non-allergic SUBJECT_SAMPLE_FACTORS P3 02_007 Classification:Non-allergic SUBJECT_SAMPLE_FACTORS P4 02_008 Classification:Non-allergic SUBJECT_SAMPLE_FACTORS P5 04_003 Classification:Non-allergic SUBJECT_SAMPLE_FACTORS P6 04_006 Classification:Non-allergic SUBJECT_SAMPLE_FACTORS P7 01_001 Classification:Mild SUBJECT_SAMPLE_FACTORS P8 01_002 Classification:Mild SUBJECT_SAMPLE_FACTORS P9 02_002 Classification:Mild SUBJECT_SAMPLE_FACTORS P10 02_003 Classification:Mild SUBJECT_SAMPLE_FACTORS P11 04_001 Classification:Mild SUBJECT_SAMPLE_FACTORS P12 01_005 Classification:Moderate SUBJECT_SAMPLE_FACTORS P13 02_001 Classification:Moderate SUBJECT_SAMPLE_FACTORS P14 02_004 Classification:Moderate SUBJECT_SAMPLE_FACTORS P15 03_004 Classification:Moderate SUBJECT_SAMPLE_FACTORS P16 03_098 Classification:Moderate SUBJECT_SAMPLE_FACTORS P17 04_002 Classification:Moderate SUBJECT_SAMPLE_FACTORS P18 03_002 Classification:Severe SUBJECT_SAMPLE_FACTORS P19 03_003 Classification:Severe SUBJECT_SAMPLE_FACTORS P20 03_092 Classification:Severe SUBJECT_SAMPLE_FACTORS P21 03_093 Classification:Severe SUBJECT_SAMPLE_FACTORS P22 03_094 Classification:Severe SUBJECT_SAMPLE_FACTORS P23 03_095 Classification:Severe SUBJECT_SAMPLE_FACTORS P24 03_096 Classification:Severe SUBJECT_SAMPLE_FACTORS P25 03_099 Classification:Severe #COLLECTION CO:COLLECTION_SUMMARY Whole blood was drawn before the profilin challenge and was divided into two CO:COLLECTION_SUMMARY tubes: one for plasma extraction and other for PBMCs isolation. Briefly, 20 ml CO:COLLECTION_SUMMARY of peripheral blood were collected to obtain plasma by centrifugation(1500rpm x CO:COLLECTION_SUMMARY 5 min.) and PBMCs using Ficoll-Paque (GE Healthcare™) density gradient CO:COLLECTION_SUMMARY centrifugation. CO:SAMPLE_TYPE Blood (whole) CO:COLLECTION_LOCATION Hospital Universitario Clínico San Carlos, Madrid, España. Hospital CO:COLLECTION_LOCATION Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa CO:COLLECTION_LOCATION (IP), Madrid, España. Hospital Virgen del Puerto, Plasencia, Cáceres, España. CO:COLLECTION_LOCATION Hospital Universitario HM Sanchinarro, Madrid, España. CO:VOLUMEORAMOUNT_COLLECTED 14-16 mL Blood CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS BD Vacutainer® Heparin Blood Collection Tubes (Ref. BD367871) CO:STORAGE_VIALS Eppendorf 1.5 mL CO:COLLECTION_TUBE_TEMP Room Temperature #TREATMENT TR:TREATMENT_SUMMARY No treatment was used in this study. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For LC-MS, plasma protein was 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 protein precipitation, 120 μL of cold SP:SAMPLEPREP_SUMMARY acetonitrile (ACN) were added to 40 μL of each plasma sample in an Eppendorf SP:SAMPLEPREP_SUMMARY and were stored on ice for 5 minutes. Then, metabolites were separated by SP:SAMPLEPREP_SUMMARY centrifugation (16,000× g, 15 min, 4 °C). From the resulting supernatant, 100 SP:SAMPLEPREP_SUMMARY μL were transferred to GC vial with insert and were evaporated to dryness SP:SAMPLEPREP_SUMMARY (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Then, 10 SP:SAMPLEPREP_SUMMARY μL of O-methoxyamine hydrochloride in pyridine (15 mg/mL) was added to each GC SP:SAMPLEPREP_SUMMARY vial, and the mixture was vigorously vortex-mixed and ultrasonicated. SP:SAMPLEPREP_SUMMARY Methoxymation was carried out in darkness, at room temperature for 16 h. SP:SAMPLEPREP_SUMMARY Afterwards, 10 μL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% SP:SAMPLEPREP_SUMMARY trimethylchlorosilane (TMCS) were added as catalyst and the solution was further SP:SAMPLEPREP_SUMMARY mixed using the vortex. For silylation process, samples were heated in an oven SP:SAMPLEPREP_SUMMARY for 1h at 70 °C. Finally, 100 μL of heptane containing 10 ppm of C18:0 methyl SP:SAMPLEPREP_SUMMARY ester (IS) were added to each GC vial and vortex-mixed before GC analysis. #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 Normal phase CH:INSTRUMENT_NAME Agilent 1200 CH:COLUMN_NAME Unspecified #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_VOLTAGE 3500 V MS:DRY_GAS_FLOW 10.5 L/min MS:DRY_GAS_TEMP 330 °C MS:FRAGMENT_VOLTAGE 175 V MS:MS_RESULTS_FILE ST000980_AN001604_Results.txt UNITS:peak area #END