#METABOLOMICS WORKBENCH hormel101_20170811_091254_mwtab.txt DATATRACK_ID:1214 STUDY_ID:ST000863 ANALYSIS_ID:AN001388 PROJECT_ID:PR000575
VERSION             	1
CREATED_ON             	August 11, 2017, 12:53 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 Street SW, Rochester, MN 55905
PR:EMAIL                         	scarisbrick.isobel@mayo.edu
PR:PHONE                         	507-284-0124
#STUDY
ST:STUDY_TITLE                   	Targeting Myelin Cholesterol in PAR1 and PAR2 Mice after SCI
ST:STUDY_SUMMARY                 	Targeting Myelin Cholesterol in PAR1 and PAR2 Mice after SCI. The samples
ST:STUDY_SUMMARY                 	submitted are purified myelin preparations from the postnatal day 21 or 60 mouse
ST:STUDY_SUMMARY                 	spinal cord (SC). There are 30 total sampes, n=5 for each group. wild type
ST:STUDY_SUMMARY                 	control, PAR1 KO and PAR2 KO at P21 and P60 days.
ST:INSTITUTE                     	Mayo Clinic
ST:LAST_NAME                     	Scarisbrick
ST:FIRST_NAME                    	Isobel
ST:ADDRESS                       	200 First Street SW, Rochester, MN 55905
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           	-	ms6120-1	Time:P60 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-2	Time:P60 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-3	Time:P60 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-10	Time:P60 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-11	Time:P60 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-4	Time:P60 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-5	Time:P60 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-12	Time:P60 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-13	Time:P60 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-6	Time:P60 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-7	Time:P60 | Group:PAR2	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-8	Time:P60 | Group:PAR2	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-9	Time:P60 | Group:PAR2	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-14	Time:P60 | Group:PAR2	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-15	Time:P60 | Group:PAR2	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-16	Time:P21 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-17	Time:P21 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-18	Time:P21 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-19	Time:P21 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-20	Time:P21 | Group:CTRL	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-21	Time:P21 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-22	Time:P21 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-23	Time:P21 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-24	Time:P21 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-25	Time:P21 | Group:PAR1	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-26	Time:P21 | Group:PAR2	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-27	Time:P21 | Group:PAR2	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-28	Time:P21 | Group:PAR2	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-29	Time:P21 | Group:PAR2	
SUBJECT_SAMPLE_FACTORS           	-	ms6120-30	Time:P21 | Group:PAR2	
#COLLECTION
CO:COLLECTION_SUMMARY            	This prior project involved frozen whole mouse spinal cord (SC) samples for
CO:COLLECTION_SUMMARY            	Untargeted Profiling (unbiased metabolomics assay). There were 9 samples total,
CO:COLLECTION_SUMMARY            	n=3 for each genotype that includes wild type control, PAR1 KO and PAR2 KO at
CO:COLLECTION_SUMMARY            	P60 days. Your team prepared the samples and ran the profiling and provided us
CO:COLLECTION_SUMMARY            	with the results. Since the outcome of this preliminary study using 3 animals
CO:COLLECTION_SUMMARY            	for each genotype looked so promising, we discussed bringing the total number to
CO:COLLECTION_SUMMARY            	5 and re-running to improve the power. Therefore, we are submitting 2 more
CO:COLLECTION_SUMMARY            	samples for each genotype at P60. In addition, we are providing 5 samples for
CO:COLLECTION_SUMMARY            	each genotype at P21 (an earlier age) for additional comparisons. To bring n=5
CO:COLLECTION_SUMMARY            	for each P60 genotype we provide 2 more samples per genotype. In addition we
CO:COLLECTION_SUMMARY            	provide n=5 for P21 for each genotype (wild type, PAR1 and PAR2).
#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            	"Cholesterol 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           	GC
CH:INSTRUMENT_NAME               	Agilent 7890A
CH:COLUMN_NAME                   	Agilent HP5-MS (30m × 0.25mm, 0.25 um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 5975C
MS:INSTRUMENT_TYPE               	Single quadrupole
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	cholesterol (ug/mg tissue)
MS_METABOLITE_DATA_START
Samples	ms6120-1	ms6120-2	ms6120-3	ms6120-10	ms6120-11	ms6120-4	ms6120-5	ms6120-12	ms6120-13	ms6120-6	ms6120-14	ms6120-15	ms6120-16	ms6120-17	ms6120-19	ms6120-20	ms6120-21	ms6120-22	ms6120-23	ms6120-24	ms6120-25	ms6120-26	ms6120-27	ms6120-28	ms6120-29
Factors	Time:P60 | Group:CTRL	Time:P60 | Group:CTRL	Time:P60 | Group:CTRL	Time:P60 | Group:CTRL	Time:P60 | Group:CTRL	Time:P60 | Group:PAR1	Time:P60 | Group:PAR1	Time:P60 | Group:PAR1	Time:P60 | Group:PAR1	Time:P60 | Group:PAR1	Time:P60 | Group:PAR2	Time:P60 | Group:PAR2	Time:P21 | Group:CTRL	Time:P21 | Group:CTRL	Time:P21 | Group:CTRL	Time:P21 | Group:CTRL	Time:P21 | Group:PAR1	Time:P21 | Group:PAR1	Time:P21 | Group:PAR1	Time:P21 | Group:PAR1	Time:P21 | Group:PAR1	Time:P21 | Group:PAR2	Time:P21 | Group:PAR2	Time:P21 | Group:PAR2	Time:P21 | Group:PAR2
Total Cholesterol	37.22	35.84	44.13	30.30	25.98	33.81	30.02	25.45	25.58	87.46	36.09	38.62	17.49	13.31	17.70	13.54	18.87	20.18	22.13	18.84	26.71	23.77	22.08	14.20	19.29
Free Cholesterol	38.72	36.57	44.50	30.20	25.54	33.63	29.80	25.33	25.43	89.85	36.07	38.05	16.79	13.24	17.78	13.30	18.78	20.12	22.06	18.59	26.63	23.57	21.84	14.19	19.03
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
Total Cholesterol
Free Cholesterol
METABOLITES_END
#END