#METABOLOMICS WORKBENCH hormel101_20170810_152538_mwtab.txt DATATRACK_ID:1211 STUDY_ID:ST000860 ANALYSIS_ID:AN001385 PROJECT_ID:PR000575 VERSION 1 CREATED_ON August 11, 2017, 12:37 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 Targeted Sphingomyelin Concentrations in Kallikrein 6 Mice after SCI ST:STUDY_SUMMARY Targeted Sphingomyelin in Kallikrein 6 Mice 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 12 samples total in Project 3, n=3 for K6+/+ or K6-/- at ST:STUDY_SUMMARY either P21 or P90. 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 - ms6026-1 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-2 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-3 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-4 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-5 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-6 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-7 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-8 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-9 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-10 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-11 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-12 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-13 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-14 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-15 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-16 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-17 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-18 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-19 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6026-20 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse #COLLECTION CO:COLLECTION_SUMMARY Tissue is from adult mouse spinal cord (SC). We are submitting these samples for CO:COLLECTION_SUMMARY Untargeted Profiling (unbiased metabolomics assay) and for lipid analysis. The CO:COLLECTION_SUMMARY lipid assays we request are 1) free fatty acid composition of lipids; 2) free CO:COLLECTION_SUMMARY fatty acid panel; 3) cholesterol concentration (free and bound); 4) Ceramides, CO:COLLECTION_SUMMARY including galactosyl and glucosyl; 5) sphingomyelin. The Untargeted profiling is CO:COLLECTION_SUMMARY our top priority, followed by the lipid assays as listed. All samples were snap CO:COLLECTION_SUMMARY frozen at the point of harvest and approximate weights are provided. The samples CO:COLLECTION_SUMMARY are submitted as intact pieces of tissue. There are two different genotypes CO:COLLECTION_SUMMARY (K6+/+ and K6-/-) and 20 samples total, n=5 for each group that includes CO:COLLECTION_SUMMARY P21(K6+/+); P90 (K6+/+); P21 (K6-/-); and P90 (K6-/-). We would like to make CO:COLLECTION_SUMMARY comparisons across the 4 groups. #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 "Sphingomyelin concentrations of mouse spinal cord Lipids will be quantified in SP:SAMPLEPREP_SUMMARY myelin 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 Quantiva QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/vial MS_METABOLITE_DATA_START Samples ms6026-1 ms6026-2 ms6026-3 ms6026-4 ms6026-5 ms6026-6 ms6026-7 ms6026-8 ms6026-9 ms6026-10 ms6026-11 ms6026-12 ms6026-13 ms6026-14 ms6026-15 ms6026-16 ms6026-17 ms6026-18 ms6026-19 ms6026-20 Factors group:K6+/+ group:K6+/+ group:K6+/+ group:K6+/+ group:K6+/+ group:K6+/+ group:K6+/+ group:K6+/+ group:K6+/+ group:K6+/+ group:K6-/- group:K6-/- group:K6-/- group:K6-/- group:K6-/- group:K6-/- group:K6-/- group:K6-/- group:K6-/- group:K6-/- SM-C16 71.4 82.1 117.5 75.8 105.7 159.5 150.9 170.4 222.9 185.2 106.3 96.0 107.3 115.6 63.2 152.8 154.6 166.8 163.3 170.6 SM-C18:1 34.3 38.6 69.6 41.3 60.9 65.6 50.4 57.3 82.8 60.6 64.2 56.1 70.5 57.5 34.4 58.4 61.8 70.1 66.8 69.1 SM-C18:0 913 1135 1820 1117 1594 2193 1865 2107 2893 2304 1582 1465 1664 1581 1001 2117 2205 2423 2366 2378 SM-C24:1 1472 1936 2842 1686 2487 4664 4256 4952 6477 5217 2084 2134 2258 2563 1833 5998 6301 6525 6851 6797 SM-C24:0 390 497 679 416 598 866 807 920 1194 1022 485 508 547 549 472 1151 1194 1219 1339 1329 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name SM-C16 SM-C18:1 SM-C18:0 SM-C24:1 SM-C24:0 METABOLITES_END #END