#METABOLOMICS WORKBENCH hormel101_20170809_154457_mwtab.txt DATATRACK_ID:1199 STUDY_ID:ST000848 ANALYSIS_ID:AN001372 PROJECT_ID:PR000575
VERSION             	1
CREATED_ON             	August 10, 2017, 3:11 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 NEFA of Kallikrein 6 Signals through PAR1 and PAR2 to Enchance
ST:STUDY_TITLE                   	Recovery of Function after SCI
ST:STUDY_SUMMARY                 	Targeting mouse myelin NEFA 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           	-	ms5959-1	time pt:P21d | grouping:CTRL	gender=Female; protein (ug/ul)=1.81
SUBJECT_SAMPLE_FACTORS           	-	ms5959-2	time pt:P21d | grouping:CTRL	gender=Female; protein (ug/ul)=1.57
SUBJECT_SAMPLE_FACTORS           	-	ms5959-3	time pt:P21d | grouping:CTRL	gender=Female; protein (ug/ul)=1.99
SUBJECT_SAMPLE_FACTORS           	-	ms5959-4	time pt:P21d | grouping:PAR1	gender=Female; protein (ug/ul)=1.42
SUBJECT_SAMPLE_FACTORS           	-	ms5959-5	time pt:P21d | grouping:PAR1	gender=Female; protein (ug/ul)=2.15
SUBJECT_SAMPLE_FACTORS           	-	ms5959-6	time pt:P21d | grouping:PAR1	gender=Female; protein (ug/ul)=1.87
SUBJECT_SAMPLE_FACTORS           	-	ms5959-7	time pt:P21d | grouping:PAR2	gender=Female; protein (ug/ul)=1.83
SUBJECT_SAMPLE_FACTORS           	-	ms5959-8	time pt:P21d | grouping:PAR2	gender=Female; protein (ug/ul)=1.21
SUBJECT_SAMPLE_FACTORS           	-	ms5959-9	time pt:P21d | grouping:PAR2	gender=Female; protein (ug/ul)=2.20
SUBJECT_SAMPLE_FACTORS           	-	ms5959-10	time pt:P60d | grouping:Wt	gender=Female; protein (ug/ul)=1.55
SUBJECT_SAMPLE_FACTORS           	-	ms5959-11	time pt:P60d | grouping:Wt	gender=Female; protein (ug/ul)=1.73
SUBJECT_SAMPLE_FACTORS           	-	ms5959-12	time pt:P60d | grouping:Wt	gender=Female; protein (ug/ul)=2.49
SUBJECT_SAMPLE_FACTORS           	-	ms5959-13	time pt:P60d | grouping:PAR2-/-	gender=Female; protein (ug/ul)=1.86
SUBJECT_SAMPLE_FACTORS           	-	ms5959-14	time pt:P60d | grouping:PAR2-/-	gender=Female; protein (ug/ul)=1.48
SUBJECT_SAMPLE_FACTORS           	-	ms5959-15	time pt:P60d | grouping:PAR2-/-	gender=Female; protein (ug/ul)=2.70
SUBJECT_SAMPLE_FACTORS           	-	ms5959-16	time pt:P60d | grouping:PAR1-/-	gender=Female; protein (ug/ul)=2.94
SUBJECT_SAMPLE_FACTORS           	-	ms5959-17	time pt:P60d | grouping:PAR1-/-	gender=Female; protein (ug/ul)=2.61
SUBJECT_SAMPLE_FACTORS           	-	ms5959-18	time pt:P60d | grouping:PAR1-/-	gender=Female; protein (ug/ul)=2.52
SUBJECT_SAMPLE_FACTORS           	-	ms5959-19	time pt:P21d | grouping:K6+/+	gender=Male; protein (ug/ul)=2.61
SUBJECT_SAMPLE_FACTORS           	-	ms5959-20	time pt:P21d | grouping:K6+/+	gender=Male; protein (ug/ul)=2.48
SUBJECT_SAMPLE_FACTORS           	-	ms5959-21	time pt:P21d | grouping:K6+/+	gender=Male; protein (ug/ul)=1.93
SUBJECT_SAMPLE_FACTORS           	-	ms5959-22	time pt:P21d | grouping:K6-/-	gender=Male; protein (ug/ul)=1.72
SUBJECT_SAMPLE_FACTORS           	-	ms5959-23	time pt:P21d | grouping:K6-/-	gender=Male; protein (ug/ul)=2.16
SUBJECT_SAMPLE_FACTORS           	-	ms5959-24	time pt:P21d | grouping:K6-/-	gender=Male; protein (ug/ul)=2.47
SUBJECT_SAMPLE_FACTORS           	-	ms5959-25	time pt:P90d | grouping:K6+/+	gender=Male; protein (ug/ul)=2.70
SUBJECT_SAMPLE_FACTORS           	-	ms5959-26	time pt:P90d | grouping:K6+/+	gender=Male; protein (ug/ul)=2.57
SUBJECT_SAMPLE_FACTORS           	-	ms5959-27	time pt:P90d | grouping:K6+/+	gender=Male; protein (ug/ul)=2.93
SUBJECT_SAMPLE_FACTORS           	-	ms5959-28	time pt:P90d | grouping:K6-/-	gender=Male; protein (ug/ul)=3.16
SUBJECT_SAMPLE_FACTORS           	-	ms5959-29	time pt:P90d | grouping:K6-/-	gender=Male; protein (ug/ul)=2.96
SUBJECT_SAMPLE_FACTORS           	-	ms5959-30	time pt:P90d | grouping:K6-/-	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            	"NEFA of mouse spinal cord Lipids will be quantified in myelin isolated in high
SP:SAMPLEPREP_SUMMARY            	yield and purity by subcellular fractionation from the lumbosacral spinal cord.
SP:SAMPLEPREP_SUMMARY            	While there are no absolutely ‘myelin-specific’ lipids, galactocerebroside
SP:SAMPLEPREP_SUMMARY            	is the most typical of myelin in the adult nervous system being directly
SP:SAMPLEPREP_SUMMARY            	proportional to the amount of myelin. Sulfatide is another galactolipid enriched
SP:SAMPLEPREP_SUMMARY            	in myelin. Together with cholesterol, these form 78% of the total amount of
SP:SAMPLEPREP_SUMMARY            	lipid in the myelin membrane and each will be quantified using LC/MS/MS. A
SP:SAMPLEPREP_SUMMARY            	highly sensitive assay for galactocerebroside was recently established by the
SP:SAMPLEPREP_SUMMARY            	Mayo Metabolomics Core and can be implemented immediately. The LC/MS/MS panel
SP:SAMPLEPREP_SUMMARY            	for free fatty acids, including the very long chain fatty acids found in myelin
SP:SAMPLEPREP_SUMMARY            	is also routinely performed by the Core. Cholesterol will be quantified using an
SP:SAMPLEPREP_SUMMARY            	NMR-based approach by the Mayo Dept. of Laboratory Medicine Clinical Core.
SP:SAMPLEPREP_SUMMARY            	Additionally, we have a plan in place with the Metabolomics Core to develop
SP:SAMPLEPREP_SUMMARY            	LC/MS/MS assays for sulfatide and sphingomyelin during the Pilot proposal.
SP:SAMPLEPREP_SUMMARY            	Having quantitative assays for each of these key myelin lipids will facilitate
SP:SAMPLEPREP_SUMMARY            	our goal to comprehensively profile myelin lipid metabolism and will form
SP:SAMPLEPREP_SUMMARY            	foundational assays for a future NIH grant focused on myelin metabolism."
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity
CH:COLUMN_NAME                   	Waters Acquity BEH C18 (150 x 2.1mm, 1.7um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6460 QQQ
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	ng/ug of protein
MS_METABOLITE_DATA_START
Samples	ms5959-1	ms5959-2	ms5959-3	ms5959-4	ms5959-5	ms5959-6	ms5959-7	ms5959-8	ms5959-9	ms5959-10	ms5959-11	ms5959-12	ms5959-13	ms5959-14	ms5959-15	ms5959-16	ms5959-17	ms5959-18	ms5959-19	ms5959-20	ms5959-21	ms5959-22	ms5959-23	ms5959-24	ms5959-25	ms5959-26	ms5959-27	ms5959-28	ms5959-29	ms5959-30
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-/-
EPA	0.17	0.17	0.18	0.23	0.20	0.18	0.16	0.13	0.16	0.13	0.11	0.07	0.07	0.08	0.06	0.08	0.09	0.11	0.10	0.13	0.12	0.16	0.15	0.17	0.06	0.07	0.06	0.05	0.06	0.08
linolenic	0.02	0.03	0.03	0.03	0.19	0.02	0.01	0.02	0.03	0.03	0.09	0.02	0.02	0.03	0.01	0.03	0.02	0.03	0.01	0.02	0.02	0.03	0.04	0.04	0.02	0.03	0.02	0.02	0.03	0.03
DHA	0.63	0.63	0.73	0.84	0.71	0.56	0.53	0.70	0.75	0.53	0.76	0.51	0.50	0.61	0.64	0.44	0.59	0.61	0.71	0.67	0.79	0.96	0.80	0.94	0.32	0.43	0.38	0.38	0.47	0.53
myristic	0.25	0.18	0.21	0.31	0.51	0.17	0.20	0.31	0.27	0.29	0.33	0.11	0.12	0.13	0.12	0.12	0.15	0.16	0.14	0.20	0.25	0.18	0.15	0.29	0.14	0.33	0.07	0.08	0.10	0.16
palmitoleic	0.24	0.23	0.27	0.33	0.29	0.24	0.19	0.24	0.26	0.16	0.22	0.15	0.14	0.17	0.14	0.11	0.15	0.12	0.19	0.22	0.27	0.31	0.25	0.34	0.07	0.12	0.09	0.07	0.08	0.19
arachidonic	5.16	4.05	4.03	6.46	4.39	5.68	3.54	3.91	4.57	3.46	3.65	3.02	2.56	2.83	2.58	3.14	3.85	3.15	3.34	3.82	4.16	4.87	4.08	5.96	2.06	2.13	2.15	1.54	1.53	2.56
palmitelaidic	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
linoleic	0.75	0.76	0.80	1.06	0.79	0.85	0.68	0.86	0.87	0.63	1.31	0.65	0.56	0.60	0.50	0.60	0.62	0.58	0.68	0.79	0.82	0.94	0.78	1.06	0.45	0.66	0.52	0.49	0.54	0.57
palmitic	4.75	5.18	4.42	6.68	6.03	5.22	4.35	5.81	4.65	7.06	7.22	5.19	5.48	6.77	5.23	3.87	4.35	3.58	4.11	4.59	5.72	6.12	4.53	7.33	3.21	4.14	3.95	3.10	3.31	4.03
oleic	7.14	7.16	6.67	9.91	6.27	8.21	6.10	8.13	6.57	7.81	8.26	6.98	6.71	8.16	6.17	5.34	6.43	5.21	5.66	6.05	7.14	8.88	7.07	8.81	4.04	4.53	4.65	3.48	3.60	5.47
elaidic	0.00	0.00	0.00	0.00	0.03	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	0.03	0.01	0.07	0.00	0.00	0.00	0.04
stearic	4.88	5.53	3.82	6.79	6.48	6.16	5.22	5.70	4.22	6.89	5.94	4.46	4.91	5.78	4.14	3.63	3.61	2.25	3.99	4.39	6.04	6.17	4.28	7.66	3.12	3.75	4.02	2.83	2.77	3.41
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
EPA
linolenic
DHA
myristic
palmitoleic
arachidonic
palmitelaidic
linoleic
palmitic
oleic
elaidic
stearic
METABOLITES_END
#END