#METABOLOMICS WORKBENCH Davidobe12_20180613_060330 DATATRACK_ID:1413 STUDY_ID:ST000980 ANALYSIS_ID:AN001606 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 GC-MS analysis was performed by a GC system (Agilent Technologies 7890A) CH:CHROMATOGRAPHY_SUMMARY equipped with an autosampler (Agilent 7693) coupled to a mass spectrometer with CH:CHROMATOGRAPHY_SUMMARY triple-Axis detector (5975C, Agilent). Two microliters (2 μL) of the CH:CHROMATOGRAPHY_SUMMARY derivatized sample were injected through a GC-Column DB5-MS (30 m length, 0.25 CH:CHROMATOGRAPHY_SUMMARY mm i.d., 0.25 μm film 95% dimethyl/5% diphenylpolysiloxane) with an integrated CH:CHROMATOGRAPHY_SUMMARY precolumn (10 m J&W, Agilent). Carrier gas (He) flow rate was set at 1 mL/min CH:CHROMATOGRAPHY_SUMMARY and injector temperature at 250 °C. Split ratio was fixed from 1:5 to 1:10 with CH:CHROMATOGRAPHY_SUMMARY 3 to 10 mL/min. He split flow into a Restek 20782 (Bellefonte, PA, USA) CH:CHROMATOGRAPHY_SUMMARY deactivated glass-wool split liner. The temperature gradient was programmed as CH:CHROMATOGRAPHY_SUMMARY follows: the initial oven temperature was set at 60 ºC (held for 1 min), CH:CHROMATOGRAPHY_SUMMARY increased to 325 ºC at 10 ºC/min rate (within 26.5 min) and hold 325 ºC for CH:CHROMATOGRAPHY_SUMMARY 10 min. The total run time was 37.5 min. A cool-down period was applied for 10 CH:CHROMATOGRAPHY_SUMMARY min before the next injection. Detector transfer line, filament source and the CH:CHROMATOGRAPHY_SUMMARY quadrupole temperature were set at 280 ºC, 230 ºC and 150 ºC, respectively. CH:CHROMATOGRAPHY_SUMMARY MS detection was performed with electron ionization (EI) mode at -70 eV. The CH:CHROMATOGRAPHY_SUMMARY mass spectrometer was operated in scan mode over a mass range of m/z 50-600 at a CH:CHROMATOGRAPHY_SUMMARY rate of 2.7 scan/s. Several IS injections, a standard mix of fatty acid methyl CH:CHROMATOGRAPHY_SUMMARY esters (FAME C8-C30), extraction blanks and 3 QCs samples were injected at the CH:CHROMATOGRAPHY_SUMMARY beginning of analysis, following QCs injections every 5 experimental samples and CH:CHROMATOGRAPHY_SUMMARY 1 QC injection at the end of worklist. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Unspecified #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 5975C MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:HELIUM_FLOW 1 mL/min MS:IONIZATION_ENERGY -70 eV #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples 02_005 02_006 02_007 02_008 04_003 04_006 01_001 01_002 02_002 02_003 04_001 01_005 02_001 02_004 03_004 03_098 04_002 03_002 03_003 03_092 03_093 03_094 03_095 03_096 03_099 Factors Classification:Non-allergic Classification:Non-allergic Classification:Non-allergic Classification:Non-allergic Classification:Non-allergic Classification:Non-allergic Classification:Mild Classification:Mild Classification:Mild Classification:Mild Classification:Mild Classification:Moderate Classification:Moderate Classification:Moderate Classification:Moderate Classification:Moderate Classification:Moderate Classification:Severe Classification:Severe Classification:Severe Classification:Severe Classification:Severe Classification:Severe Classification:Severe Classification:Severe Adenosine 0 0 591 559 364 0 0 0 2191 441 0 2240 0 762 3754 3650 2050 809 0 1495 3642 1492 4418 2457 3462 Aminoisobutyric acid 19532 18228 21362 12403 35259 8828 11543 7828 14226 8395 13542 29434 19891 23679 16373 22148 31351 13051 56627 16844 20199 23105 18033 3511 24474 Aminomalonic acid-2TMS 10498 8055 10082 4444 17727 4950 5962 3388 7309 4044 6315 14387 9875 10173 7787 10686 13813 7488 25922 7189 10998 9749 8360 2132 10918 Aminomalonic acid-3TMS 5453 6622 5744 6338 9216 4581 7006 4926 7669 5203 6514 6660 12583 7271 6017 9649 11137 7676 9491 4586 6185 6322 7056 7238 9337 Cysteine 19531 10077 22046 19265 10390 9921 4207 8136 7886 7788 7517 9618 12881 11350 21784 13915 6621 11497 9838 7087 4908 7606 10049 7897 11038 D-Allose 1311783 1450009 1424805 1337644 1566724 2038419 2095826 1558144 1287608 1419181 1209437 1582695 1602446 1870016 1316074 882479 1714644 1074901 1474066 1237038 867825 1003398 470854 600268 1427434 D-Glucose/D-Altrose/D-Galactose /Talose 9559741 10815559 10411011 9604849 11076620 12956630 13985394 11559423 9522013 10418088 8827265 11483709 11656721 11314360 9224615 6462066 11729149 8155238 10882109 8768221 6338256 6961355 3441760 4051634 10398069 D-Lyxosylamine 3872 2966 3034 3096 4517 3107 3404 3017 3815 2872 5930 2391 2174 3136 2340 2114 2772 2680 8271 3288 2856 2730 2746 3408 3131 D-Mannose /D-Allose 58384 48153 51449 67245 62027 77412 87402 69808 64538 75776 52518 45367 76332 53668 52982 36982 52582 39348 40712 52962 38383 53874 18892 29648 45192 D-sorbitol 575 1495 1498 2613 1184 944 3516 2028 1544 1551 44627 3082 1761 2480 2656 11834 1434 1677 3059 18704 1417 1803 3485 2331 5418 Fructose 16089 18160 19233 15462 24102 12890 57803 7796 24848 11155 51883 13935 21318 13405 10534 9205 19074 11570 23286 13735 6633 6701 11409 9600 10399 Glyceric acid 13017 10218 11717 12214 6867 7088 4025 7189 6322 7388 7174 5314 7194 8112 14788 8980 4297 10478 5254 9700 8066 8380 10315 9950 8906 Hypoxanthine 12427 8683 11048 14952 13559 687 5116 12246 13948 11467 16471 8247 15062 17042 45544 36604 11334 121848 11310 40289 31909 36244 38070 79343 40908 Lactamide 80068 140873 152511 108546 131246 183046 120939 126957 129264 114911 92362 107617 292357 111159 116459 161957 189519 132738 142882 150431 157112 134439 137790 116028 137184 Lactic acid 7177139 7548771 7283038 10494308 10674209 9298276 8858358 8516391 11802485 13954089 11614404 7446156 10384592 8642101 12673937 13134694 7887101 15110467 9778145 18114265 12078806 9644456 13678492 15556728 15756381 Lauric acid 2440 2497 3348 3997 1390 8331 6569 1273 2491 1407 1133 330 3923 704 1051 1167 1683 2267 1859 749 2060 2603 1777 4250 1877 Leucine 203058 130236 353367 645233 332551 10409 403398 771467 468578 808113 471194 353538 314491 504604 454085 314764 278844 471135 286583 396423 121173 285348 564662 689166 473680 L-Sorbose/Fructose 17133 15473 14927 19069 15697 20828 22156 17275 22391 21897 14269 14339 21183 17182 14792 12050 12736 11135 11728 15070 10361 15473 6461 7564 15081 Methionine 12202 11096 7162 5130 11453 4561 8325 6126 8132 4816 11316 14258 9553 11829 9355 10525 10143 8528 14418 13131 14461 13955 10661 11115 10937 Methyl-b-D-Galactopyranoside 122462 374954 346207 246415 193500 238682 248105 309012 184002 329460 205647 200160 240415 134138 187074 151210 140721 282513 170960 315759 511040 235378 246397 328458 178851 Myo-inositol 46881 31112 38117 37502 41083 43186 48176 43146 46377 56030 47521 33361 38602 50964 74508 56186 38030 44224 46842 35239 48975 40559 34274 15049 49522 Myristic acid 14490 18092 20089 14330 9458 12729 13465 7583 10525 7661 13959 3161 8864 6618 8528 6721 19569 16498 7511 11874 10873 11026 8157 11902 8657 Oxalic acid 541502 1034682 1098426 695855 857019 1417556 842669 862384 837386 755883 589891 703672 2128680 777256 786950 1121312 1440557 880557 1014915 957830 1102835 992222 889567 749720 871342 Palmitic acid 142867 122547 248051 263565 143096 275286 147556 107455 185821 135667 109463 119207 146593 143302 168943 178476 151888 302234 127381 153136 173801 207716 209747 268902 183298 Phenylalanine 67804 24200 73111 100264 76160 11258 81913 163939 115122 113544 95341 67730 41350 91538 88679 70947 68320 79371 93822 55787 62061 72021 74515 68271 62029 Pyruvic acid 54149 39822 70535 76602 106799 127684 344700 75080 145554 128474 112262 75080 103736 78772 22280 53616 93476 42595 69407 86546 52480 22728 58755 63033 37829 Serine 67652 76019 43610 30350 70809 56923 35237 22092 40239 25418 45444 70855 40130 51116 43670 67816 50044 45011 77787 68870 89877 51532 36814 23765 40926 Stearic acid 45488 39685 60358 67032 47150 55038 40117 29240 50091 36126 26263 33858 48283 31071 42127 46583 45222 69405 33057 42894 41007 48791 53244 70770 41724 Tagatose 11043 12131 13341 10019 18534 8877 43873 7580 17697 7920 39001 9671 12110 8729 7266 5453 14750 6370 16322 9740 5522 5372 7776 6336 8299 Talose 679695 726962 723655 674254 779875 1012732 1037046 804780 657642 713734 612849 800388 815897 916765 646934 462114 865765 570599 757113 637902 452243 493806 243442 296737 731788 Trihydroxybutyric acid 41667 20530 40868 34938 17240 20129 6033 12092 13938 12733 11981 18609 21010 18512 41548 24566 12616 17466 18176 12549 9634 14273 20086 16249 23325 Urea 9804890 10223697 7029211 9457047 5076651 3907944 8687310 7952770 6772929 5979573 9616029 9681414 8254317 6042890 10405722 7647956 12413274 9333798 9060552 11112832 8464560 10642882 11605817 8464560 14435687 Valine 275493 230093 535731 852016 505632 33440 624784 1120593 683802 1071440 789346 485098 515830 701347 621798 495790 532228 616114 463182 510661 186047 419618 764785 787778 662225 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID HMDB ID CAS_ID Adenosine 23.825 236 58-61-7 Aminoisobutyric acid 12.457 218 10569-71-9 Aminomalonic acid-2TMS 11.87 218 73168-02-2 Aminomalonic acid-3TMS 12.474 320 1000079-52-5 Cysteine 13.582 220 52-90-4 D-Allose 17.521 319 579-36-2 D-Glucose/D-Altrose/D-Galactose /Talose 17.284 319 59-23-4 D-Lyxosylamine 14.861 103 39840-37-4 D-Mannose /D-Allose 17.216 319 87-78-5 D-sorbitol 17.898 319 50-70-4 Fructose 17.288 217 57-48-7 Glyceric acid 10.735 189 473-81-4 Hypoxanthine 16.442 265 68-94-0 Lactamide 7.959 100 2043-43-8 Lactic acid 6.851 117 79-33-4 Lauric acid 14.789 117 143-07-7 Leucine 8.298 86 61-90-5 L-Sorbose/Fructose 17.235 103 3615-56-3 Methionine 11.835 61 63-68-3 Methyl-b-D-Galactopyranoside 16.935 217 1824-94-8 Myo-inositol 19.354 217 87-89-8 Myristic acid 16.887 285 544-63-8 Oxalic acid 7.883 147 144-62-7 Palmitic acid 18.846 117 64519-82-0 Phenylalanine 13.545 120 63-91-2 Pyruvic acid 6.714 174 127-17-3 Serine 9.706 116 56-45-1 Stearic acid 20.675 341 57-11-4 Tagatose 17.011 103 87-81-0 Talose 17.584 103 2595-98-4 Trihydroxybutyric acid 13.575 292 38191-88-7 Urea 9.599 189 57-13-6 Valine 7.296 72 72-18-4 METABOLITES_END #END