#METABOLOMICS WORKBENCH albertofalco_20250211_103709 DATATRACK_ID:5617 STUDY_ID:ST003781 ANALYSIS_ID:AN006209 PROJECT_ID:PR002359
VERSION                          	1
CREATED_ON                       	05-23-2025
#PROJECT
PR:PROJECT_TITLE                 	Metabolic Therapies for the Treatment of Infectious Diseases in Farmed Fish
PR:PROJECT_TITLE                 	(MetDisFish)
PR:PROJECT_TYPE                  	MS quantitative analysis
PR:PROJECT_SUMMARY               	To identify metabolic processes and/or metabolites that may influence greater or
PR:PROJECT_SUMMARY               	lesser resistance to infectious diseases in farmed fish, in order to design new
PR:PROJECT_SUMMARY               	metabolic therapies aimed at reducing the impact of these diseases in
PR:PROJECT_SUMMARY               	aquaculture.
PR:INSTITUTE                     	Consejo Superior de Investigaciones Científicas
PR:DEPARTMENT                    	Institute of Aquaculture Torre de la Sal (IATS-CSIC)
PR:LAST_NAME                     	Falco
PR:FIRST_NAME                    	Alberto
PR:ADDRESS                       	Torre de la Sal sn, Ribera de Cabanes, Castellón, 12595, Spain
PR:EMAIL                         	alberto.falco@csic.es
PR:PHONE                         	0034964187477
PR:FUNDING_SOURCE                	The European Maritime, Fisheries and Aquaculture Fund (EMFAF) and the Ministry
PR:FUNDING_SOURCE                	of Agriculture, Fisheries and Food (MAPA) [grant number MetDisFish]
PR:PUBLICATIONS                  	https://doi.org/10.1016/j.aquaculture.2025.742721
PR:DOI                           	http://dx.doi.org/10.21228/M8Q538
PR:CONTRIBUTORS                  	Patricia Pereiro, Alberto Falcó, Marta Fernández-Oliver, Rocío Paladea-Rojo,
PR:CONTRIBUTORS                  	Raúl Bonet-García, José E. Yuste, Magalí Rey-Campos, Antonio Figueras,
PR:CONTRIBUTORS                  	Ricardo Mallavia, Beatriz Novoa
#STUDY
ST:STUDY_TITLE                   	Metabolomics Study in Turbot on Susceptibility to Bacterial Infections
ST:STUDY_TYPE                    	MS quantitative analysis
ST:STUDY_SUMMARY                 	In the present study it was conducted an untargeted metabolome analysis of the
ST:STUDY_SUMMARY                 	head kidney, the primary hematopoietic tissue in fish, and the liver, the main
ST:STUDY_SUMMARY                 	metabolic organ, from two full-sibling turbot (Scophthalmus maximus) families
ST:STUDY_SUMMARY                 	that exhibit different susceptibility to the pathogenic bacterium Aeromonas
ST:STUDY_SUMMARY                 	salmonicida subsp. salmonicida. This Gram-negative bacterium is the etiologic
ST:STUDY_SUMMARY                 	agent of classical furunculosis, a widespread disease that poses severe
ST:STUDY_SUMMARY                 	challenges for the aquaculture of various salmonid species (Dallaire-Dufresne et
ST:STUDY_SUMMARY                 	al., 2014 [10.1016/j.vetmic.2013.06.025]), as well as for turbot (Toranzo and
ST:STUDY_SUMMARY                 	Barja, 1992 [Bull. Eur. Ass. Fish. Pathol. 12(5):147-149]; Pedersen and Larsen,
ST:STUDY_SUMMARY                 	1996 [Bull. Eur. Ass. Fish. Pathol. 16, 129–133]), a species of considerable
ST:STUDY_SUMMARY                 	economic importance in both European and Chinese aquaculture
ST:STUDY_SUMMARY                 	(Fernández-González et al., 2023 [10.1111/raq.12747]). These metabolomics
ST:STUDY_SUMMARY                 	analyses allowed us to identify several metabolites potentially involved in
ST:STUDY_SUMMARY                 	resistance due to their differential abundance in the resistant family. Briefly,
ST:STUDY_SUMMARY                 	after minor manual quality filtering, 158 significant features were identified
ST:STUDY_SUMMARY                 	for all liver comparisons in positive mode and 32 in negative mode. Similarly,
ST:STUDY_SUMMARY                 	185 significant features were retrieved for all head kidney comparisons in
ST:STUDY_SUMMARY                 	positive mode and 25 in negative mode after discarding high masses (≥800 Da).
ST:STUDY_SUMMARY                 	The identification of annotated features was performed using CEU Mass Mediator
ST:STUDY_SUMMARY                 	3.0, followed by a comparison of the fragmentation patterns. Thus, a total of 18
ST:STUDY_SUMMARY                 	features, with molecular weights ranging from 116 to 613 Da, were found to be
ST:STUDY_SUMMARY                 	annotated from all the significant features previously retrieved in the liver
ST:STUDY_SUMMARY                 	comparisons. Among them, various compounds related to amino acids and
ST:STUDY_SUMMARY                 	derivatives were identified, including L-gamma-glutamyl-L-cysteine, L-tyrosine,
ST:STUDY_SUMMARY                 	L-asparagine, gamma-L-glutamyl-L-2-aminobutyrate, S-adenosyl-L-methionine, and
ST:STUDY_SUMMARY                 	L-proline. Compounds related to nitrogenous bases such as guanosine
ST:STUDY_SUMMARY                 	monophosphate, uridine, hypoxanthine, cytidine monophosphate and its neuraminic
ST:STUDY_SUMMARY                 	derivative, adenylsuccinic acid, and adenine were also obtained. Among the
ST:STUDY_SUMMARY                 	significant features observed in the head kidney comparisons, there were
ST:STUDY_SUMMARY                 	annotated 11 with molecular weights ranging from 89 to 489 Da. They comprised
ST:STUDY_SUMMARY                 	various organic acids, including malic and succinic acids, as well as amino
ST:STUDY_SUMMARY                 	acids and derivatives, such as 5-L-glutamyl-taurine and L-glutamic acid.
ST:INSTITUTE                     	Consejo Superior de Investigaciones Científicas
ST:DEPARTMENT                    	Institute of Aquaculture Torre de la Sal (IATS-CSIC)
ST:LAST_NAME                     	Falco
ST:FIRST_NAME                    	Alberto
ST:ADDRESS                       	IATS-CSIC, Torre de la Sal sn, Ribera de Cabanes, Castellón, 12595, Spain
ST:EMAIL                         	alberto.falco@csic.es
ST:PHONE                         	0034964187477
ST:SUBMIT_DATE                   	2025-02-11
#SUBJECT
SU:SUBJECT_TYPE                  	Fish
SU:SUBJECT_SPECIES               	Scophthalmus maximus
SU:TAXONOMY_ID                   	52904
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	16907	Sample source:Head kidney | Treatment:Control | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M13_33035_C HK-1_2-C,5_01_16907.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16908	Sample source:Head kidney | Treatment:Control | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M14_33035_C HK-2_2-C,6_01_16908.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16909	Sample source:Head kidney | Treatment:Control | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M15_33035_C HK-3_2-C,7_01_16909.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16944	Sample source:Head kidney | Treatment:Control | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M13_33035_C HK-1_2-C,5_01_16944.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16945	Sample source:Head kidney | Treatment:Control | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M14_33035_C HK-2_2-C,6_01_16945.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16946	Sample source:Head kidney | Treatment:Control | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M15_33035_C HK-3_2-C,7_01_16946.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16910	Sample source:Head kidney | Treatment:Control | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M16_33039_C HK-1_2-C,8_01_16910.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16911	Sample source:Head kidney | Treatment:Control | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M17_33039_C HK-2_2-D,1_01_16911.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16912	Sample source:Head kidney | Treatment:Control | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M18_33039_C HK-3_2-D,2_01_16912.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16947	Sample source:Head kidney | Treatment:Control | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M16_33039_C HK-1_2-C,8_01_16947.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16948	Sample source:Head kidney | Treatment:Control | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M17_33039_C HK-2_2-D,1_01_16948.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16949	Sample source:Head kidney | Treatment:Control | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M18_33039_C HK-3_2-D,2_01_16949.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16914	Sample source:Head kidney | Treatment:Infected | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M19_33035_BAC HK-1_2-D,3_01_16914.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16915	Sample source:Head kidney | Treatment:Infected | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M20_33035_BAC HK-2_2-D,4_01_16915.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16916	Sample source:Head kidney | Treatment:Infected | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M21_33035_BAC HK-3_2-D,5_01_16916.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16951	Sample source:Head kidney | Treatment:Infected | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M19_33035_BAC HK-1_2-D,3_01_16951.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16952	Sample source:Head kidney | Treatment:Infected | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M20_33035_BAC HK-2_2-D,4_01_16952.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16953	Sample source:Head kidney | Treatment:Infected | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M21_33035_BAC HK-3_2-D,5_01_16953.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16917	Sample source:Head kidney | Treatment:Infected | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M22_33039_BAC HK-1_2-D,6_01_16917.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16918	Sample source:Head kidney | Treatment:Infected | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M23_33039_BAC HK-2_2-D,7_01_16918.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16919	Sample source:Head kidney | Treatment:Infected | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M24_33039_BAC HK-3_2-D,8_01_16919.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16954	Sample source:Head kidney | Treatment:Infected | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M22_33039_BAC HK-1_2-D,6_01_16954.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16955	Sample source:Head kidney | Treatment:Infected | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M23_33039_BAC HK-2_2-D,7_01_16955.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16956	Sample source:Head kidney | Treatment:Infected | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M24_33039_BAC HK-3_2-D,8_01_16956.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16906	Sample source:Head kidney | Treatment:Solvent | Genotype:None	Ion polarity=Positive; RAW_FILE_NAME=QC2_HK_+MS_2-F,8_01_16906.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16943	Sample source:Head kidney | Treatment:Solvent | Genotype:None	Ion polarity=Negative; RAW_FILE_NAME=QC2_HK_-MS_2-F,8_01_16943.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16892	Sample source:Liver | Treatment:Control | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M1_33035_C LIV-1_2-B,1_01_16892.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16893	Sample source:Liver | Treatment:Control | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M2_33035_C LIV-2_2-B,2_01_16893.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16894	Sample source:Liver | Treatment:Control | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M3_33035_C LIV-3_2-B,3_01_16894.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16929	Sample source:Liver | Treatment:Control | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M1_33035_C LIV-1_2-B,1_01_16929.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16930	Sample source:Liver | Treatment:Control | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M2_33035_C LIV-2_2-B,2_01_16930.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16931	Sample source:Liver | Treatment:Control | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M3_33035_C LIV-3_2-B,3_01_16931.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16895	Sample source:Liver | Treatment:Control | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M4_33039_C LIV-1_2-B,4_01_16895.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16896	Sample source:Liver | Treatment:Control | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M5_33039_C LIV-2_2-B,5_01_16896.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16897	Sample source:Liver | Treatment:Control | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M6_33039_C LIV-3_2-B,6_01_16897.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16932	Sample source:Liver | Treatment:Control | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M4_33039_C LIV-1_2-B,4_01_16932.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16933	Sample source:Liver | Treatment:Control | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M5_33039_C LIV-2_2-B,5_01_16933.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16934	Sample source:Liver | Treatment:Control | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M6_33039_C LIV-3_2-B,6_01_16934.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16899	Sample source:Liver | Treatment:Infected | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M7_33035_BAC LIV-1_2-B,7_01_16899.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16900	Sample source:Liver | Treatment:Infected | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M8_33035_BAC LIV-2_2-B,8_01_16900.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16901	Sample source:Liver | Treatment:Infected | Genotype:Resistant	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M9_33035_BAC LIV-3_2-C,1_01_16901.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16936	Sample source:Liver | Treatment:Infected | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M7_33035_BAC LIV-1_2-B,7_01_16936.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16937	Sample source:Liver | Treatment:Infected | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M8_33035_BAC LIV-2_2-B,8_01_16937.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16938	Sample source:Liver | Treatment:Infected | Genotype:Resistant	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M9_33035_BAC LIV-3_2-C,1_01_16938.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16902	Sample source:Liver | Treatment:Infected | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M10_33039_BAC LIV-1_2-C,2_01_16902.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16903	Sample source:Liver | Treatment:Infected | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M11_33039_BAC LIV-2_2-C,3_01_16903.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16904	Sample source:Liver | Treatment:Infected | Genotype:Susceptible	Ion polarity=Positive; RAW_FILE_NAME=MET1136-M12_33039_BAC LIV-3_2-C,4_01_16904.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16939	Sample source:Liver | Treatment:Infected | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M10_33039_BAC LIV-1_2-C,2_01_16939.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16940	Sample source:Liver | Treatment:Infected | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M11_33039_BAC LIV-2_2-C,3_01_16940.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16941	Sample source:Liver | Treatment:Infected | Genotype:Susceptible	Ion polarity=Negative; RAW_FILE_NAME=MET1136-M12_33039_BAC LIV-3_2-C,4_01_16941.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16891	Sample source:Liver | Treatment:Solvent | Genotype:None	Ion polarity=Positive; RAW_FILE_NAME=QC1_LIV_+MS_2-F,1_01_16891.mzXML
SUBJECT_SAMPLE_FACTORS           	-	16925	Sample source:Liver | Treatment:Solvent | Genotype:None	Ion polarity=Negative; RAW_FILE_NAME=QC1_LIV_-MS_2-F,1_01_16925.mzXML
#COLLECTION
CO:COLLECTION_SUMMARY            	Five full-sibling turbot families (initial body weight 8-9 g) of the exact same
CO:COLLECTION_SUMMARY            	age (identified as 22035, 22037, 22038, 22039 and 22040) were used to evaluate
CO:COLLECTION_SUMMARY            	their susceptibility to an A. salmonicida subsp. salmonicida intraperitoneal
CO:COLLECTION_SUMMARY            	(i.p.) challenge (5.9 × 108 CFU/mL, volume 100 µL) as we previously described
CO:COLLECTION_SUMMARY            	(Pereiro et al., 2024 [10.3389/fimmu.2024.1522666]). We found that family 22035
CO:COLLECTION_SUMMARY            	showed a significantly higher survival rate compared to the other four families,
CO:COLLECTION_SUMMARY            	and being family 22039 the most susceptible to the bacteria, being considered as
CO:COLLECTION_SUMMARY            	resistant and susceptible families, respectively (Pereiro et al., 2024
CO:COLLECTION_SUMMARY            	[10.3389/fimmu.2024.1522666]). At 24 hours post-infection (hpi) we took head
CO:COLLECTION_SUMMARY            	kidney and liver samples from uninfected (inoculated with 100 µL of PBS) and
CO:COLLECTION_SUMMARY            	infected fish belonging to each family (n=15) for metabolome analysis. The same
CO:COLLECTION_SUMMARY            	quantity of tissue from 5 fish was pooled to obtain 3 biological replicates (5
CO:COLLECTION_SUMMARY            	fish/replicate). Four experimental groups per tissue were obtained and named as
CO:COLLECTION_SUMMARY            	resistant control (RC), resistant infected (RI), susceptible control (SC) and
CO:COLLECTION_SUMMARY            	susceptible infected (SI). The weight of each pool was determined and a volume
CO:COLLECTION_SUMMARY            	of 4 µL of cold CHCl3:MeOH (20:30 v/v) per mg of tissue was added to each tube.
CO:COLLECTION_SUMMARY            	The samples, maintained in ice, were disrupted with a potter-type homogenizer
CO:COLLECTION_SUMMARY            	until completely disaggregated. Extracts were centrifuged (Eppendorf 5415R,
CO:COLLECTION_SUMMARY            	Hamburg, Germany) for 20 min at 13,000 rpm and 4°C, and the supernatants
CO:COLLECTION_SUMMARY            	containing both polar and non-polar metabolites were transferred to a clean tube
CO:COLLECTION_SUMMARY            	and maintained at -80°C until untargeted metabolomics analysis.
CO:SAMPLE_TYPE                   	Heart kidney, Liver
#TREATMENT
TR:TREATMENT_SUMMARY             	Aeromonas salmonicida subsp. salmonicida intraperitoneal (i.p.) challenge (5.9
TR:TREATMENT_SUMMARY             	× 108 CFU/mL, volume 100 µL) as we previously described (Pereiro et al., 2024
TR:TREATMENT_SUMMARY             	[10.3389/fimmu.2024.1522666]). Control fish were injected with 100 ul of PBS.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The weight of each pool was determined and a volume of 4 µL of cold CHCl3:MeOH
SP:SAMPLEPREP_SUMMARY            	(20:30 v/v) per mg of tissue was added to each tube. The samples, maintained in
SP:SAMPLEPREP_SUMMARY            	ice, were disrupted with a potter-type homogenizer until completely
SP:SAMPLEPREP_SUMMARY            	disaggregated. Extracts were centrifuged (Eppendorf 5415R, Hamburg, Germany) for
SP:SAMPLEPREP_SUMMARY            	20 min at 13,000 rpm and 4°C, and the supernatants containing both polar and
SP:SAMPLEPREP_SUMMARY            	non-polar metabolites were transferred to a clean tube and maintained at -80°C
SP:SAMPLEPREP_SUMMARY            	until untargeted metabolomics analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Separation was performed at 30°C using a reverse phase Poroshell 120 EC-C18
CH:CHROMATOGRAPHY_SUMMARY        	column (3.0 x 100 mm, 2.7 µm particle size) (Agilent Technologies, Waldbronn,
CH:CHROMATOGRAPHY_SUMMARY        	Germany). The mobile phases consisted of acidified water (phase A) and acidified
CH:CHROMATOGRAPHY_SUMMARY        	acetonitrile (phase B), both containing 0.1% v/v formic acid. The elution
CH:CHROMATOGRAPHY_SUMMARY        	gradient was as follows: 1-18% B in 0-10 min, 18-38% B in 10-16 min, 38-95% B in
CH:CHROMATOGRAPHY_SUMMARY        	16-22 min, 95-1% B in 22-23 min and 1% B in 23-28.5 min. The flow rate was 0.4
CH:CHROMATOGRAPHY_SUMMARY        	mL/min, and 3 µL of the sample was injected using a flow-through needle (FTN)
CH:CHROMATOGRAPHY_SUMMARY        	injection with a 15 µL needle. The sample compartment in the autosampler was
CH:CHROMATOGRAPHY_SUMMARY        	maintained at 6°C.
CH:INSTRUMENT_NAME               	ACQUITY I-Class system
CH:COLUMN_NAME                   	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
CH:COLUMN_TEMPERATURE            	30
CH:FLOW_GRADIENT                 	All linear: 1-18% B in 0-10 min, 18-38% B in 10-16 min, 38-95% B in 16-22 min,
CH:FLOW_GRADIENT                 	95-1% B in 22-23 min and 1% B in 23-28.5 min
CH:FLOW_RATE                     	0.4 mL/min
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile; 0.1% formic acid
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Bruker maXis Impact qTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	Mass spectrometry was performed using HR-TOF-MS in positive electrospray
MS:MS_COMMENTS                   	ionisation mode using broadband Collision Induced Dissociation (bbCID). The
MS:MS_COMMENTS                   	mass-to-charge (m/z) range was 50–1,200. ESI analyses were performed with
MS:MS_COMMENTS                   	capillary and end-plate offset voltages of -4,500 and -500 V in positive mode.
MS:MS_COMMENTS                   	Nitrogen was used as both nebulizer and drying gas. The nebulizer gas pressure
MS:MS_COMMENTS                   	was 2.0 bar, the drying gas temperature was 200 °C and its flow rate 9.0 L/min.
MS:MS_COMMENTS                   	High- and low-collision energy data were collected simultaneously by alternating
MS:MS_COMMENTS                   	the acquisition between MS and bbCID conditions (MS/MS). At the beginning of
MS:MS_COMMENTS                   	each UPLC run, a 10 mM sodium formate solution in 1:1 isopropanol-water was
MS:MS_COMMENTS                   	introduced into the ESI source for calibration of the mass axis using a divert
MS:MS_COMMENTS                   	valve. The external calibrant solution was delivered by a KNAUER Smartline Pump
MS:MS_COMMENTS                   	100 with a pressure sensor (KNAUER, Berlin, Germany). The software used for
MS:MS_COMMENTS                   	HR-TOF-MS acquisition, analysis and instrument control was oTOFcontrol 3.2
MS:MS_COMMENTS                   	(Bruker Daltonics) and HyStar 3.2 (Bruker Daltonics). DataAnalysis 4.1 software
MS:MS_COMMENTS                   	was used for post-run mass calibration and MS and MS/MS data processing. The
MS:MS_COMMENTS                   	data were converted to mzXML format for further processing.
MS:ION_MODE                      	POSITIVE
MS:MS_RESULTS_FILE               	ST003781_AN006209_Results.txt	UNITS:Intensity units	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END