#METABOLOMICS WORKBENCH hormel101_20170810_081024_mwtab.txt DATATRACK_ID:1200 STUDY_ID:ST000849 ANALYSIS_ID:AN001373 PROJECT_ID:PR000575 VERSION 1 CREATED_ON August 10, 2017, 3:20 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 FFA Compostion of Kallikrein 6 Signals through PAR1 and PAR2 to ST:STUDY_TITLE Enchance Recovery of Function after SCI ST:STUDY_SUMMARY Targeting mouse myelin FFA composition of Kallikrein 6 Signals through PAR1 and ST:STUDY_SUMMARY PAR2 to Enchance Recovery of Function after SCI. The samples submitted are ST:STUDY_SUMMARY purified myelin preparations from the postnatal day 21, 60, or 90 mouse spinal ST:STUDY_SUMMARY cord (SC). There are 9 samples total in Project 1, n=3 for each genotype ST:STUDY_SUMMARY (control, PAR1 or PAR2) at 21 days. There are 9 samples total in Project 2, n=3 ST:STUDY_SUMMARY for each genotype (WT, PAR1-/- or PAR2-/-) at 60 days. There are 12 samples ST:STUDY_SUMMARY total in Project 3, 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 - ms5960-1 time pt:P21d | grouping:CTRL gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-2 time pt:P21d | grouping:CTRL gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-3 time pt:P21d | grouping:CTRL gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-4 time pt:P21d | grouping:PAR1 gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-5 time pt:P21d | grouping:PAR1 gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-6 time pt:P21d | grouping:PAR1 gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-7 time pt:P21d | grouping:PAR2 gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-8 time pt:P21d | grouping:PAR2 gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-9 time pt:P21d | grouping:PAR2 gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-10 time pt:P60d | grouping:Wt gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-11 time pt:P60d | grouping:Wt gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-12 time pt:P60d | grouping:Wt gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-13 time pt:P60d | grouping:PAR2-/- gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-14 time pt:P60d | grouping:PAR2-/- gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-15 time pt:P60d | grouping:PAR2-/- gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-16 time pt:P60d | grouping:PAR1-/- gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-17 time pt:P60d | grouping:PAR1-/- gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-18 time pt:P60d | grouping:PAR1-/- gender=Female SUBJECT_SAMPLE_FACTORS - ms5960-19 time pt:P21d | grouping:K6+/+ gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-20 time pt:P21d | grouping:K6+/+ gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-21 time pt:P21d | grouping:K6+/+ gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-22 time pt:P21d | grouping:K6-/- gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-23 time pt:P21d | grouping:K6-/- gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-24 time pt:P21d | grouping:K6-/- gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-25 time pt:P90d | grouping:K6+/+ gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-26 time pt:P90d | grouping:K6+/+ gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-27 time pt:P90d | grouping:K6+/+ gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-28 time pt:P90d | grouping:K6-/- gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-29 time pt:P90d | grouping:K6-/- gender=Male SUBJECT_SAMPLE_FACTORS - ms5960-30 time pt:P90d | grouping:K6-/- gender=Male #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 "FFA composition of mouse spinal cord Lipids will be quantified in myelin SP:SAMPLEPREP_SUMMARY isolated in high yield and purity by subcellular fractionation from the SP:SAMPLEPREP_SUMMARY lumbosacral spinal cord. While there are no absolutely ‘myelin-specific’ SP:SAMPLEPREP_SUMMARY lipids, galactocerebroside is the most typical of myelin in the adult nervous SP:SAMPLEPREP_SUMMARY system 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 NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS % total MS_METABOLITE_DATA_START Samples ms5960-1 ms5960-2 ms5960-3 ms5960-4 ms5960-5 ms5960-6 ms5960-7 ms5960-8 ms5960-9 ms5960-10 ms5960-11 ms5960-12 ms5960-13 ms5960-14 ms5960-15 ms5960-16 ms5960-17 ms5960-18 ms5960-19 ms5960-20 ms5960-21 ms5960-22 ms5960-23 ms5960-24 ms5960-25 ms5960-26 ms5960-27 ms5960-28 ms5960-29 ms5960-30 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 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 linolenic 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 DHA 0.4 0.4 0.4 0.5 0.5 0.4 0.5 0.6 0.5 0.2 0.3 0.3 0.3 0.4 0.4 0.3 0.3 0.2 0.6 0.4 0.4 0.5 0.5 0.5 0.3 0.2 0.2 0.3 0.3 0.3 myristic 0.6 0.6 0.6 0.7 0.6 0.6 0.7 0.6 0.6 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.6 0.6 0.6 0.6 0.5 0.6 0.2 0.3 0.4 0.3 0.3 0.2 palmitoleic 2.3 2.2 2.2 2.7 0.0 0.0 0.0 0.0 2.3 1.0 0.0 0.0 1.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.2 2.2 2.2 0.0 0.9 0.0 0.9 0.9 0.0 0.9 arachidonic 2.1 2.1 2.0 2.3 2.2 2.1 2.2 2.2 2.2 1.9 2.0 2.0 2.0 2.2 2.0 1.8 1.9 1.9 2.3 2.1 2.2 2.2 2.1 2.2 1.8 1.8 1.8 1.9 1.8 1.9 palmitelaidic 0.0 0.0 0.0 0.0 2.4 2.3 2.4 2.5 0.0 0.0 1.1 1.1 0.0 1.2 1.1 1.1 1.2 1.0 2.4 2.4 0.0 0.0 0.0 2.2 0.0 1.0 0.8 0.0 0.9 0.0 linoleic 1.1 1.0 1.0 1.3 1.2 1.1 1.3 1.3 1.1 0.4 0.5 0.4 0.5 0.5 0.4 0.4 0.5 0.4 1.2 1.2 1.2 1.1 1.0 1.1 0.3 0.4 0.3 0.3 0.3 0.4 palmitic 14.3 15.1 14.5 17.9 15.9 15.9 17.2 15.8 15.6 12.6 11.4 12.0 14.2 12.9 12.7 12.1 12.3 11.5 14.9 15.2 15.3 16.4 14.8 15.4 12.0 13.2 12.5 13.4 13.1 12.7 oleic 68.0 66.1 67.4 59.8 64.6 64.4 62.5 64.8 64.7 69.8 73.9 71.3 68.5 69.9 70.9 71.4 71.3 73.5 66.9 65.4 63.5 62.5 62.6 64.3 70.9 67.8 69.1 67.4 67.5 69.4 elaidic 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 stearic 11.2 12.6 12.0 14.7 12.6 13.2 13.4 12.1 13.0 13.7 10.4 12.6 13.0 12.6 12.3 12.5 12.2 11.2 11.2 12.7 14.6 14.5 16.3 13.7 13.5 15.3 14.0 15.4 15.8 14.1 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name EPA linolenic DHA myristic palmitoleic arachidonic palmitelaidic linoleic palmitic oleic elaidic stearic METABOLITES_END #END