#METABOLOMICS WORKBENCH yorch40_20210601_033941 DATATRACK_ID:2662 STUDY_ID:ST001810 ANALYSIS_ID:AN002933 PROJECT_ID:PR001144
VERSION             	1
CREATED_ON             	June 1, 2021, 7:48 am
#PROJECT
PR:PROJECT_TITLE                 	Metabolomics-driven identification of biochemical mechanisms underlying the
PR:PROJECT_TITLE                 	neuroprotective effects of pleiotrophin in a mouse model of Parkinson’s
PR:PROJECT_TITLE                 	disease
PR:PROJECT_SUMMARY               	Pleiotrophin (PTN) is a cytokine involved in nerve tissue repair processes,
PR:PROJECT_SUMMARY               	neuroinflammation and neuronal survival. PTN expression levels are upregulated
PR:PROJECT_SUMMARY               	in the nigrostriatal pathway of Parkinson’s Disease (PD) patients. We aimed to
PR:PROJECT_SUMMARY               	characterize the dopaminergic injury and glial activation in the nigrostriatal
PR:PROJECT_SUMMARY               	pathway of mice with transgenic Ptn overexpression in the brain (Ptn-Tg) after
PR:PROJECT_SUMMARY               	intrastriatal injection of the Parkinsonian toxin 6-hydroxydopamine (6-OHDA).
PR:PROJECT_SUMMARY               	The injection of 6-OHDA induced a significant decrease of the number of tyrosine
PR:PROJECT_SUMMARY               	hydroxylase (TH)-positive neurons in the substantia nigra and of the striatal TH
PR:PROJECT_SUMMARY               	contents in Wild type (Wt) mice. In contrast, these effects of 6-OHDA were
PR:PROJECT_SUMMARY               	blocked in Ptn-Tg mice. 6-OHDA injection did not cause robust changes in
PR:PROJECT_SUMMARY               	microglia but induced an exacerbated astrocytic response in Wt mice compared
PR:PROJECT_SUMMARY               	with Ptn-Tg mice. In metabolomics studies, we detected interesting metabolites
PR:PROJECT_SUMMARY               	that significantly discriminate the more injured 6-OHDA-injected Wt striatum and
PR:PROJECT_SUMMARY               	the more protected 6-OHDA-injected Ptn-Tg striatum. Particularly, we detected
PR:PROJECT_SUMMARY               	groups of metabolites, mostly corresponding to phospholipids, whose trends were
PR:PROJECT_SUMMARY               	opposite in both groups. In summary, the data confirm the neuroprotective effect
PR:PROJECT_SUMMARY               	of brain PTN overexpression in this mouse model of PD. New lipid-related PD drug
PR:PROJECT_SUMMARY               	candidates emerge from this study and the data presented here support the
PR:PROJECT_SUMMARY               	increasingly recognized “lipid cascade” in PD.
PR:INSTITUTE                     	CEU San Pablo University
PR:LAST_NAME                     	Sáiz
PR:FIRST_NAME                    	Jorge
PR:ADDRESS                       	JULIÁN ROMEA 23, Madrid, Madrid, 28003, Spain
PR:EMAIL                         	jorge.saizgalindo@ceu.es
PR:PHONE                         	913 72 47 11
#STUDY
ST:STUDY_TITLE                   	Metabolomics-driven identification of biochemical mechanisms underlying the
ST:STUDY_TITLE                   	neuroprotective effects of pleiotrophin in a mouse model of Parkinson’s
ST:STUDY_TITLE                   	disease
ST:STUDY_SUMMARY                 	Pleiotrophin (PTN) is a cytokine involved in nerve tissue repair processes,
ST:STUDY_SUMMARY                 	neuroinflammation and neuronal survival. PTN expression levels are upregulated
ST:STUDY_SUMMARY                 	in the nigrostriatal pathway of Parkinson’s Disease (PD) patients. We aimed to
ST:STUDY_SUMMARY                 	characterize the dopaminergic injury and glial activation in the nigrostriatal
ST:STUDY_SUMMARY                 	pathway of mice with transgenic Ptn overexpression in the brain (Ptn-Tg) after
ST:STUDY_SUMMARY                 	intrastriatal injection of the Parkinsonian toxin 6-hydroxydopamine (6-OHDA).
ST:STUDY_SUMMARY                 	The injection of 6-OHDA induced a significant decrease of the number of tyrosine
ST:STUDY_SUMMARY                 	hydroxylase (TH)-positive neurons in the substantia nigra and of the striatal TH
ST:STUDY_SUMMARY                 	contents in Wild type (Wt) mice. In contrast, these effects of 6-OHDA were
ST:STUDY_SUMMARY                 	blocked in Ptn-Tg mice. 6-OHDA injection did not cause robust changes in
ST:STUDY_SUMMARY                 	microglia but induced an exacerbated astrocytic response in Wt mice compared
ST:STUDY_SUMMARY                 	with Ptn-Tg mice. In metabolomics studies, we detected interesting metabolites
ST:STUDY_SUMMARY                 	that significantly discriminate the more injured 6-OHDA-injected Wt striatum and
ST:STUDY_SUMMARY                 	the more protected 6-OHDA-injected Ptn-Tg striatum. Particularly, we detected
ST:STUDY_SUMMARY                 	groups of metabolites, mostly corresponding to phospholipids, whose trends were
ST:STUDY_SUMMARY                 	opposite in both groups. In summary, the data confirm the neuroprotective effect
ST:STUDY_SUMMARY                 	of brain PTN overexpression in this mouse model of PD. New lipid-related PD drug
ST:STUDY_SUMMARY                 	candidates emerge from this study and the data presented here support the
ST:STUDY_SUMMARY                 	increasingly recognized “lipid cascade” in PD.
ST:INSTITUTE                     	CEMBIO
ST:LAST_NAME                     	Sáiz
ST:FIRST_NAME                    	Jorge
ST:ADDRESS                       	JULIÁN ROMEA 23, Madrid, Madrid, 28003, Spain
ST:EMAIL                         	jorge.saizgalindo@ceu.es
ST:PHONE                         	none
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	WT1-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT1-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT2-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT2-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT3-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT3-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT4-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT4-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT5-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT5-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT6-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT6-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT7-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT7-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT8-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT8-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT9-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT9-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT10-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT10-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT11-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT11-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT12-HD-POS.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT12-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T1-HD-POS.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T1-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T2-HD-POS.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T2-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T3-HD-POS.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T3-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T4-HD-POS.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T4-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T5-HD-POS.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T5-HD-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT1-VEH-POS.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT1-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT2-VEH-POS.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT2-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT3-VEH-POS.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT3-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT4-VEH-POS.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT4-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT6-VEH-POS.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT6-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT7-VEH-POS.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT7-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT8-VEH-POS.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT8-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT9-VEH-POS.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT9-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT10-VEH-POS.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT10-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T1-VEH-POS.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T1-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T2-VEH-POS.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T2-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T4-VEH-POS.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T4-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T5-VEH-POS.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T5-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T6-VEH-POS.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T6-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	T7-VEH-POS.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T7-VEH-POS.d; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	QC1-POS	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC1-POS; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	QC2-POS	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC2-POS; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	QC3-POS	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC3-POS; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	QC4-POS	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC4-POS; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	QC5-POS	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC5-POS; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	QC6-POS	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC6-POS; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	QC7-POS	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC7-POS; Polarity=POS
SUBJECT_SAMPLE_FACTORS           	-	WT1-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT1-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT2-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT2-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT3-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT3-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT4-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT4-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT5-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT5-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT6-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT6-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT7-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT7-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT8-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT8-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT9-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT9-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT10-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT10-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT11-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT11-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT12-HD-NEG.d	Subject type:WT | Treatment:OH-Dopamine	RAW_FILE_NAME=WT12-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T1-HD-NEG.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T1-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T2-HD-NEG.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T2-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T3-HD-NEG.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T3-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T4-HD-NEG.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T4-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T5-HD-NEG.d	Subject type:Transg | Treatment:OH-Dopamine	RAW_FILE_NAME=T5-HD-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT1-VEH-NEG.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT1-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT2-VEH-NEG.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT2-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT3-VEH-NEG.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT3-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT4-VEH-NEG.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT4-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT6-VEH-NEG.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT6-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT7-VEH-NEG.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT7-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT8-VEH-NEG.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT8-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT9-VEH-NEG.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT9-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	WT10-VEH-NEG.d	Subject type:WT | Treatment:Vehicle	RAW_FILE_NAME=WT10-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T1-VEH-NEG.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T1-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T2-VEH-NEG.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T2-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T4-VEH-NEG.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T4-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T5-VEH-NEG.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T5-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T6-VEH-NEG.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T6-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	T7-VEH-NEG.d	Subject type:Transg | Treatment:Vehicle	RAW_FILE_NAME=T7-VEH-NEG.d; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	QC1-NEG	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC1-NEG; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	QC2-NEG	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC2-NEG; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	QC3-NEG	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC3-NEG; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	QC4-NEG	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC4-NEG; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	QC5-NEG	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC5-NEG; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	QC6-NEG	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC6-NEG; Polarity=NEG
SUBJECT_SAMPLE_FACTORS           	-	QC7-NEG	Subject type:QC | Treatment:QC	RAW_FILE_NAME=QC7-NEG; Polarity=NEG
#COLLECTION
CO:COLLECTION_SUMMARY            	Samples were collected and frozen at -80ºC
CO:SAMPLE_TYPE                   	Brain
#TREATMENT
TR:TREATMENT_SUMMARY             	Striatum resections were added to 300 µL MeOH:water (50:50) in 2 mL Eppendorf
TR:TREATMENT_SUMMARY             	tubes and were first homogenized with glass beads in a TissueLyser LT (QIAGEN)
TR:TREATMENT_SUMMARY             	for 2 min. The tubes were immersed in liquid N2 and homogenized again in the
TR:TREATMENT_SUMMARY             	TissueLyser LT for another 2 min. A volume 100 µL of the homogenate was
TR:TREATMENT_SUMMARY             	transferred into Eppendorf tubes of 1.5 mL and added with 320 µL of methanol
TR:TREATMENT_SUMMARY             	and 80 µL of MTBE and the mixture was vortexed for 1 h. Afterwards, the vials
TR:TREATMENT_SUMMARY             	were centrifuged at 4000 g for 20 min at 20 ºC and 300 µL of the supernatants
TR:TREATMENT_SUMMARY             	were transferred into new tubes, which were evaporated to dryness in a vacuum
TR:TREATMENT_SUMMARY             	concentrator. Finally, the residues were reconstituted in 50 µL of
TR:TREATMENT_SUMMARY             	MeOH:water:MTBE (37:5:4), being the samples ready for their analysis. Blank
TR:TREATMENT_SUMMARY             	samples were prepared following the sample procedure without the addition of any
TR:TREATMENT_SUMMARY             	biological tissue.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Striatum resections were added to 300 µL MeOH:water (50:50) in 2 mL Eppendorf
SP:SAMPLEPREP_SUMMARY            	tubes and were first homogenized with glass beads in a TissueLyser LT (QIAGEN)
SP:SAMPLEPREP_SUMMARY            	for 2 min. The tubes were immersed in liquid N2 and homogenized again in the
SP:SAMPLEPREP_SUMMARY            	TissueLyser LT for another 2 min. A volume 100 µL of the homogenate was
SP:SAMPLEPREP_SUMMARY            	transferred into Eppendorf tubes of 1.5 mL and added with 320 µL of methanol
SP:SAMPLEPREP_SUMMARY            	and 80 µL of MTBE and the mixture was vortexed for 1 h. Afterwards, the vials
SP:SAMPLEPREP_SUMMARY            	were centrifuged at 4000 g for 20 min at 20 ºC and 300 µL of the supernatants
SP:SAMPLEPREP_SUMMARY            	were transferred into new tubes, which were evaporated to dryness in a vacuum
SP:SAMPLEPREP_SUMMARY            	concentrator. Finally, the residues were reconstituted in 50 µL of
SP:SAMPLEPREP_SUMMARY            	MeOH:water:MTBE (37:5:4), being the samples ready for their analysis. Blank
SP:SAMPLEPREP_SUMMARY            	samples were prepared following the sample procedure without the addition of any
SP:SAMPLEPREP_SUMMARY            	biological tissue.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1200
CH:COLUMN_NAME                   	RP C8 column Agilent Poroshell (150 mm 2.1 mm, 2.7 μm)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6520 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	All data were controlled and acquired using Mass Hunter Qualitative Analysis
MS:MS_COMMENTS                   	B.07.00 (Agilent Technologies). Data obtained from LC-MS were cleaned of
MS:MS_COMMENTS                   	background noise and unrelated ions. Peak detection, deconvolution and alignment
MS:MS_COMMENTS                   	were performed by the recursive feature extraction (RFE) using Profinder
MS:MS_COMMENTS                   	Software B.08.00 (Agilent Technologies). Blank subtraction and filtering by
MS:MS_COMMENTS                   	frequency of at least 50% of the QC and 60% of each group and relative standard
MS:MS_COMMENTS                   	deviation (RSD) less than 30% in QC were performed, to keep only the relevant
MS:MS_COMMENTS                   	features. Missing values were substituted by KNN algorithm.
MS:MS_RESULTS_FILE               	ST001810_AN002933_Results.txt	UNITS:aera	Has m/z:Neutral masses	Has RT:Yes	RT units:Minutes
#END