#METABOLOMICS WORKBENCH hormel101_20170810_094107_mwtab.txt DATATRACK_ID:1202 STUDY_ID:ST000851 ANALYSIS_ID:AN001375 PROJECT_ID:PR000575
VERSION             	1
CREATED_ON             	August 10, 2017, 3:26 pm
#PROJECT
PR:PROJECT_TITLE                 	Mayo Pilot and Feasiblity: Targeting Myelin Metabolism of Kallikrein 6 Signals
PR:PROJECT_TITLE                 	through PAR1 and PAR2 to Enchance Recovery of Function after SCI
PR:PROJECT_SUMMARY               	CNS trauma generates a proteolytic imbalance contributing to secondary injury,
PR:PROJECT_SUMMARY               	including axonopathy and neuron degeneration. Kallikrein 6 (Klk6) is a serine
PR:PROJECT_SUMMARY               	protease implicated in neurodegeneration and here we investigate the role of
PR:PROJECT_SUMMARY               	protease activated receptors 1 (PAR1) and PAR2 in mediating these effects in
PR:PROJECT_SUMMARY               	mice with spinal cord injury (SCI). This project conducts untargeted Profiling
PR:PROJECT_SUMMARY               	(unbiased metabolomics assay) and targeted lipid analysis. The lipid assays are
PR:PROJECT_SUMMARY               	1) free fatty acid composition of lipids; 2) free fatty acid panel; 3)
PR:PROJECT_SUMMARY               	cholesterol concentration (free and bound); 4) Ceramides, including galactosyl
PR:PROJECT_SUMMARY               	and glucosyl; 5) sphingomyelin.
PR:INSTITUTE                     	Mayo Clinic
PR:LAST_NAME                     	Scarisbrick
PR:FIRST_NAME                    	Isobel
PR:ADDRESS                       	200 First St. SW, Rochester, Minnesota, 55905, USA
PR:EMAIL                         	scarisbrick.isobel@mayo.edu
PR:PHONE                         	507-284-0124
#STUDY
ST:STUDY_TITLE                   	Targeting Myelin Sphingolipids of Kallikrein 6 Signals through PAR1 and PAR2 to
ST:STUDY_TITLE                   	Enchance Recovery of Function after SCI
ST:STUDY_SUMMARY                 	Targeting Myelin Sphingolipids of Kallikrein 6 Signals through PAR1 and PAR2 to
ST:STUDY_SUMMARY                 	Enchance Recovery of Function after SCI. The samples submitted are purified
ST:STUDY_SUMMARY                 	myelin preparations from the postnatal day 21, 60, or 90 mouse spinal cord (SC).
ST:STUDY_SUMMARY                 	There are 9 samples total in Project 1, n=3 for each genotype (control, PAR1 or
ST:STUDY_SUMMARY                 	PAR2) at 21 days. There are 9 samples total in Project 2, n=3 for each genotype
ST:STUDY_SUMMARY                 	(WT, PAR1-/- or PAR2-/-) at 60 days. There are 12 samples total in Project 3,
ST:STUDY_SUMMARY                 	n=3 for K6+/+ or K6-/- at either P21 or P90.
ST:INSTITUTE                     	Mayo Clinic
ST:LAST_NAME                     	Scarisbrick
ST:FIRST_NAME                    	Isobel
ST:ADDRESS                       	200 First St. SW, Rochester, Minnesota, 55905, USA
ST:EMAIL                         	scarisbrick.isobel@mayo.edu
ST:PHONE                         	507-284-0124
#SUBJECT
SU:SUBJECT_TYPE                  	Mouse
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	ms5962-1 (12/17/15)	time pt:P21d | grouping:CTRL	study date=12/2/2015; gender=Female; protein (ug/ul)=1.81
SUBJECT_SAMPLE_FACTORS           	-	ms5962-2 (12/17/15)	time pt:P21d | grouping:CTRL	study date=12/3/2015; gender=Female; protein (ug/ul)=1.57
SUBJECT_SAMPLE_FACTORS           	-	ms5962-3 (12/17/15)	time pt:P21d | grouping:CTRL	study date=12/3/2015; gender=Female; protein (ug/ul)=1.99
SUBJECT_SAMPLE_FACTORS           	-	ms5962-4 (12/17/15)	time pt:P21d | grouping:PAR1	study date=11/17/2015; gender=Female; protein (ug/ul)=1.42
SUBJECT_SAMPLE_FACTORS           	-	ms5962-5 (12/17/15)	time pt:P21d | grouping:PAR1	study date=12/2/2015; gender=Female; protein (ug/ul)=2.15
SUBJECT_SAMPLE_FACTORS           	-	ms5962-6 (12/17/15)	time pt:P21d | grouping:PAR1	study date=12/3/2015; gender=Female; protein (ug/ul)=1.87
SUBJECT_SAMPLE_FACTORS           	-	ms5962-7 (12/17/15)	time pt:P21d | grouping:PAR2	study date=11/19/2015; gender=Female; protein (ug/ul)=1.83
SUBJECT_SAMPLE_FACTORS           	-	ms5962-8 (12/17/15)	time pt:P21d | grouping:PAR2	study date=11/19/2015; gender=Female; protein (ug/ul)=1.21
SUBJECT_SAMPLE_FACTORS           	-	ms5962-9 (12/17/15)	time pt:P21d | grouping:PAR2	study date=11/17/2015; gender=Female; protein (ug/ul)=2.2
SUBJECT_SAMPLE_FACTORS           	-	ms5962-1 (1/21/16)	time pt:P60d | grouping:Wt	study date=1/21/2016; gender=Female; protein (ug/ul)=1.55
SUBJECT_SAMPLE_FACTORS           	-	ms5962-2 (1/21/16)	time pt:P60d | grouping:Wt	study date=1/21/2016; gender=Female; protein (ug/ul)=1.73
SUBJECT_SAMPLE_FACTORS           	-	ms5962-3 (1/21/16)	time pt:P60d | grouping:Wt	study date=1/21/2016; gender=Female; protein (ug/ul)=2.49
SUBJECT_SAMPLE_FACTORS           	-	ms5962-4 (1/21/16)	time pt:P60d | grouping:PAR2-/-	study date=1/21/2016; gender=Female; protein (ug/ul)=1.86
SUBJECT_SAMPLE_FACTORS           	-	ms5962-5 (1/21/16)	time pt:P60d | grouping:PAR2-/-	study date=1/21/2016; gender=Female; protein (ug/ul)=1.48
SUBJECT_SAMPLE_FACTORS           	-	ms5962-6 (1/21/16)	time pt:P60d | grouping:PAR2-/-	study date=1/21/2016; gender=Female; protein (ug/ul)=2.7
SUBJECT_SAMPLE_FACTORS           	-	ms5962-7 (1/21/16)	time pt:P60d | grouping:PAR1-/-	study date=1/21/2016; gender=Female; protein (ug/ul)=2.94
SUBJECT_SAMPLE_FACTORS           	-	ms5962-8 (1/21/16)	time pt:P60d | grouping:PAR1-/-	study date=1/21/2016; gender=Female; protein (ug/ul)=2.61
SUBJECT_SAMPLE_FACTORS           	-	ms5962-9 (1/21/16)	time pt:P60d | grouping:PAR1-/-	study date=1/21/2016; gender=Female; protein (ug/ul)=2.52
SUBJECT_SAMPLE_FACTORS           	-	ms5962-10 (1/21/16)	time pt:P21d | grouping:K6+/+	study date=12/9/2015; gender=Male; protein (ug/ul)=2.61
SUBJECT_SAMPLE_FACTORS           	-	ms5962-11 (1/21/16)	time pt:P21d | grouping:K6+/+	study date=12/9/2015; gender=Male; protein (ug/ul)=2.48
SUBJECT_SAMPLE_FACTORS           	-	ms5962-12 (1/21/16)	time pt:P21d | grouping:K6+/+	study date=12/16/2015; gender=Male; protein (ug/ul)=1.93
SUBJECT_SAMPLE_FACTORS           	-	ms5962-13 (1/21/16)	time pt:P21d | grouping:K6-/-	study date=12/9/2015; gender=Male; protein (ug/ul)=1.72
SUBJECT_SAMPLE_FACTORS           	-	ms5962-14 (1/21/16)	time pt:P21d | grouping:K6-/-	study date=12/9/2015; gender=Male; protein (ug/ul)=2.16
SUBJECT_SAMPLE_FACTORS           	-	ms5962-15 (1/21/16)	time pt:P21d | grouping:K6-/-	study date=12/16/2015; gender=Male; protein (ug/ul)=2.47
SUBJECT_SAMPLE_FACTORS           	-	ms5962-16 (1/21/16)	time pt:P90d | grouping:K6+/+	study date=12/11/2015; gender=Male; protein (ug/ul)=2.7
SUBJECT_SAMPLE_FACTORS           	-	ms5962-17 (1/21/16)	time pt:P90d | grouping:K6+/+	study date=12/11/2015; gender=Male; protein (ug/ul)=2.57
SUBJECT_SAMPLE_FACTORS           	-	ms5962-18 (1/21/16)	time pt:P90d | grouping:K6+/+	study date=12/16/2015; gender=Male; protein (ug/ul)=2.93
SUBJECT_SAMPLE_FACTORS           	-	ms5962-19 (1/21/16)	time pt:P90d | grouping:K6-/-	study date=12/11/2015; gender=Male; protein (ug/ul)=3.16
SUBJECT_SAMPLE_FACTORS           	-	ms5962-20 (1/21/16)	time pt:P90d | grouping:K6-/-	study date=12/11/2015; gender=Male; protein (ug/ul)=2.96
SUBJECT_SAMPLE_FACTORS           	-	ms5962-21 (1/21/16)	time pt:P90d | grouping:K6-/-	study date=12/16/2015; gender=Male; protein (ug/ul)=3.88
#COLLECTION
CO:COLLECTION_SUMMARY            	The samples submitted are purified myelin preparations from the postnatal day 21
CO:COLLECTION_SUMMARY            	or 90 mouse spinal cord (SC). There are two projects but these can be run
CO:COLLECTION_SUMMARY            	together since the down stream assays are identical. There are 9 samples total
CO:COLLECTION_SUMMARY            	in Project 1, n=3 for each genotype (WT, PAR1-/- or PAR2-/-). There are 12
CO:COLLECTION_SUMMARY            	samples total in Project 2, n=3 for K6+/+ or K6-/- at either P21 or P90. We are
CO:COLLECTION_SUMMARY            	submitting these samples for analysis of 1) free fatty acid panel; 2) free fatty
CO:COLLECTION_SUMMARY            	acid composition of lipids; 3) cholesterol concentration (free and bound); 4)
CO:COLLECTION_SUMMARY            	Ceramides, including galactosyl and glucosyl; 5) sphingomyelin. A total of
CO:COLLECTION_SUMMARY            	approximately 130 to 150 ul for each sample is submitted with the protein
CO:COLLECTION_SUMMARY            	concentrations already measured by BCA assay and indicated below. Concentrations
CO:COLLECTION_SUMMARY            	are provided in ug/ul and the total volume is also indicated. One tube for each
CO:COLLECTION_SUMMARY            	sample is submitted but this can be shared across the assays.
#TREATMENT
TR:TREATMENT_SUMMARY             	A 3g Clip produces moderate SCI including demyelination and clinical impairment
TR:TREATMENT_SUMMARY             	and we recently published a detailed methodology. At 1 week after injury, the 3g
TR:TREATMENT_SUMMARY             	injured mice are expected to have an average Basso Mouse Scale score (BMS)=5 on
TR:TREATMENT_SUMMARY             	a 9 point scale such that they have frequent plantar stepping with no or some
TR:TREATMENT_SUMMARY             	coordination. This level of impairment was chosen to provide a sufficient window
TR:TREATMENT_SUMMARY             	to observe recovery.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	"Sphingolipids of mouse spinal cord Lipids will be quantified in myelin isolated
SP:SAMPLEPREP_SUMMARY            	in high yield and purity by subcellular fractionation from the lumbosacral
SP:SAMPLEPREP_SUMMARY            	spinal cord. While there are no absolutely ‘myelin-specific’ lipids,
SP:SAMPLEPREP_SUMMARY            	galactocerebroside is the most typical of myelin in the adult nervous system
SP:SAMPLEPREP_SUMMARY            	being directly proportional to the amount of myelin. Sulfatide is another
SP:SAMPLEPREP_SUMMARY            	galactolipid enriched in myelin. Together with cholesterol, these form 78% of
SP:SAMPLEPREP_SUMMARY            	the total amount of lipid in the myelin membrane and each will be quantified
SP:SAMPLEPREP_SUMMARY            	using LC/MS/MS. A highly sensitive assay for galactocerebroside was recently
SP:SAMPLEPREP_SUMMARY            	established by the Mayo Metabolomics Core and can be implemented immediately.
SP:SAMPLEPREP_SUMMARY            	The LC/MS/MS panel for free fatty acids, including the very long chain fatty
SP:SAMPLEPREP_SUMMARY            	acids found in myelin is also routinely performed by the Core. Cholesterol will
SP:SAMPLEPREP_SUMMARY            	be quantified using an NMR-based approach by the Mayo Dept. of Laboratory
SP:SAMPLEPREP_SUMMARY            	Medicine Clinical Core. Additionally, we have a plan in place with the
SP:SAMPLEPREP_SUMMARY            	Metabolomics Core to develop LC/MS/MS assays for sulfatide and sphingomyelin
SP:SAMPLEPREP_SUMMARY            	during the Pilot proposal. Having quantitative assays for each of these key
SP:SAMPLEPREP_SUMMARY            	myelin lipids will facilitate our goal to comprehensively profile myelin lipid
SP:SAMPLEPREP_SUMMARY            	metabolism and will form foundational assays for a future NIH grant focused on
SP:SAMPLEPREP_SUMMARY            	myelin metabolism."
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters Acquity BEH C8 (150 x 2mm, 1.7um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Quantum Ultra
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	unit = ng per ug of protein
MS_METABOLITE_DATA_START
Samples	ms5962-1 (12/17/15)	ms5962-2 (12/17/15)	ms5962-3 (12/17/15)	ms5962-4 (12/17/15)	ms5962-5 (12/17/15)	ms5962-6 (12/17/15)	ms5962-7 (12/17/15)	ms5962-8 (12/17/15)	ms5962-9 (12/17/15)	ms5962-1 (1/21/16)	ms5962-2 (1/21/16)	ms5962-3 (1/21/16)	ms5962-4 (1/21/16)	ms5962-5 (1/21/16)	ms5962-6 (1/21/16)	ms5962-7 (1/21/16)	ms5962-8 (1/21/16)	ms5962-9 (1/21/16)	ms5962-10 (1/21/16)	ms5962-11 (1/21/16)	ms5962-12 (1/21/16)	ms5962-13 (1/21/16)	ms5962-14 (1/21/16)	ms5962-15 (1/21/16)	ms5962-16 (1/21/16)	ms5962-17 (1/21/16)	ms5962-18 (1/21/16)	ms5962-19 (1/21/16)	ms5962-20 (1/21/16)	ms5962-21 (1/21/16)
Factors	time pt:P21d | grouping:CTRL	time pt:P21d | grouping:CTRL	time pt:P21d | grouping:CTRL	time pt:P21d | grouping:PAR1	time pt:P21d | grouping:PAR1	time pt:P21d | grouping:PAR1	time pt:P21d | grouping:PAR2	time pt:P21d | grouping:PAR2	time pt:P21d | grouping:PAR2	time pt:P60d | grouping:Wt	time pt:P60d | grouping:Wt	time pt:P60d | grouping:Wt	time pt:P60d | grouping:PAR2-/-	time pt:P60d | grouping:PAR2-/-	time pt:P60d | grouping:PAR2-/-	time pt:P60d | grouping:PAR1-/-	time pt:P60d | grouping:PAR1-/-	time pt:P60d | grouping:PAR1-/-	time pt:P21d | grouping:K6+/+	time pt:P21d | grouping:K6+/+	time pt:P21d | grouping:K6+/+	time pt:P21d | grouping:K6-/-	time pt:P21d | grouping:K6-/-	time pt:P21d | grouping:K6-/-	time pt:P90d | grouping:K6+/+	time pt:P90d | grouping:K6+/+	time pt:P90d | grouping:K6+/+	time pt:P90d | grouping:K6-/-	time pt:P90d | grouping:K6-/-	time pt:P90d | grouping:K6-/-
Sph	0.09	0.09	0.11	0.10	0.09	0.08	0.11	0.13	0.10	0.09	0.10	0.11	0.11	0.10	0.11	0.08	0.10	0.11	0.08	0.09	0.09	0.11	0.09	0.11	0.06	0.09	0.08	0.13	0.11	0.10
SPA	0.06	0.06	0.07	0.04	0.05	0.05	0.06	0.08	0.05	0.02	0.02	0.03	0.03	0.02	0.03	0.03	0.03	0.03	0.06	0.05	0.05	0.06	0.05	0.07	0.02	0.02	0.02	0.03	0.03	0.03
S1P	0.02	0.02	0.03	0.03	0.02	0.01	0.02	0.03	0.03	0.01	0.03	0.02	0.02	0.03	0.03	0.01	0.04	0.03	0.02	0.02	0.02	0.01	0.02	0.01	0.01	0.02	0.01	0.02	0.03	0.01
C8-cer	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
C14-cer	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.01	0.01	0.01	0.01	0.01	0.00	0.00	0.00	0.00	0.00	0.00
C16-cer	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.01	0.01	0.01	0.01	0.03	0.02	0.01	0.01	0.01	0.02	0.02	0.02	0.02	0.02	0.02	0.01	0.01	0.01	0.01	0.01	0.01
C16-dh cer	0.01	0.02	0.01	0.00	0.01	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.01	0.00	0.00	0.00	0.00	0.00	0.00	0.00
C18:1-cer	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
C18:1-dh cer	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
C18-cer	3.00	2.91	2.56	2.76	2.54	2.53	2.68	2.42	2.22	1.92	1.92	1.51	2.05	2.85	2.53	1.86	1.79	1.66	2.45	2.28	2.32	2.85	2.75	2.91	1.06	0.91	0.87	1.10	1.07	1.33
C18-dh cer	0.54	0.56	0.47	0.21	0.35	0.39	0.30	0.32	0.35	0.10	0.10	0.08	0.11	0.09	0.10	0.14	0.12	0.16	0.29	0.37	0.28	0.38	0.39	0.46	0.05	0.06	0.04	0.06	0.04	0.05
C20-cer	1.14	1.03	1.05	0.73	0.82	0.89	0.92	0.76	0.90	0.48	0.55	0.46	0.57	0.72	0.62	0.57	0.58	0.66	0.64	0.82	0.66	0.95	0.94	0.93	0.33	0.32	0.33	0.33	0.34	0.36
C22-cer	4.03	4.53	4.25	4.37	3.44	4.29	4.38	3.44	4.47	3.03	3.20	2.67	3.75	4.28	3.36	2.64	2.78	3.21	2.72	3.74	3.86	4.38	4.14	3.49	1.28	1.35	1.37	1.59	1.47	1.48
C24:1-cer	4.72	4.56	4.72	4.62	4.07	4.04	4.48	3.83	4.72	4.66	5.03	4.27	4.30	5.23	4.38	3.92	4.51	4.43	3.62	4.13	4.01	4.45	4.16	4.23	2.49	2.61	2.84	2.83	2.71	2.63
C24-cer	1.50	1.37	1.39	1.53	1.39	1.29	1.80	1.24	1.58	0.75	0.85	0.67	0.86	1.03	0.86	0.64	0.68	0.81	0.92	1.27	0.99	1.39	1.30	1.15	0.34	0.33	0.33	0.36	0.36	0.35
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
Sph
SPA
S1P
C8-cer
C14-cer
C16-cer
C16-dh cer
C18:1-cer
C18:1-dh cer
C18-cer
C18-dh cer
C20-cer
C22-cer
C24:1-cer
C24-cer
METABOLITES_END
#END