#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