#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