#METABOLOMICS WORKBENCH hormel101_20170810_145238_mwtab.txt DATATRACK_ID:1209 STUDY_ID:ST000858 ANALYSIS_ID:AN001383 PROJECT_ID:PR000575 VERSION 1 CREATED_ON August 11, 2017, 12:31 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 Sphingolipids in Kallikrein 6 Mice after SCI ST:STUDY_SUMMARY Targeted Sphingolipids 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 - ms6024-1 timept:P21 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-2 timept:P21 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-3 timept:P21 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-4 timept:P21 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-5 timept:P21 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-6 timept:P90 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-7 timept:P90 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-8 timept:P90 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-9 timept:P90 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-10 timept:P90 | Grouping:K6+/+ gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-11 timept:P21 | Grouping:K6-/- gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-12 timept:P21 | Grouping:K6-/- gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-13 timept:P21 | Grouping:K6-/- gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-14 timept:P21 | Grouping:K6-/- gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-15 timept:P21 | Grouping:K6-/- gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-16 timept:P90 | Grouping:K6-/- gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-17 timept:P90 | Grouping:K6-/- gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-18 timept:P90 | Grouping:K6-/- gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-19 timept:P90 | Grouping:K6-/- gender=male; Sample type=spinal cord; Species=mouse SUBJECT_SAMPLE_FACTORS - ms6024-20 timept:P90 | Grouping:K6-/- gender=male; Sample type=spinal cord; 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 "Sphingolipids 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 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 POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS units = ng/vial MS_METABOLITE_DATA_START Samples ms6024-1 ms6024-2 ms6024-3 ms6024-4 ms6024-5 ms6024-6 ms6024-7 ms6024-8 ms6024-9 ms6024-10 ms6024-11 ms6024-12 ms6024-13 ms6024-14 ms6024-15 ms6024-16 ms6024-17 ms6024-18 ms6024-19 ms6024-20 Factors timept:P21 | Grouping:K6+/+ timept:P21 | Grouping:K6+/+ timept:P21 | Grouping:K6+/+ timept:P21 | Grouping:K6+/+ timept:P21 | Grouping:K6+/+ timept:P90 | Grouping:K6+/+ timept:P90 | Grouping:K6+/+ timept:P90 | Grouping:K6+/+ timept:P90 | Grouping:K6+/+ timept:P90 | Grouping:K6+/+ timept:P21 | Grouping:K6-/- timept:P21 | Grouping:K6-/- timept:P21 | Grouping:K6-/- timept:P21 | Grouping:K6-/- timept:P21 | Grouping:K6-/- timept:P90 | Grouping:K6-/- timept:P90 | Grouping:K6-/- timept:P90 | Grouping:K6-/- timept:P90 | Grouping:K6-/- timept:P90 | Grouping:K6-/- Sph 1.53 1.34 2.12 1.37 1.61 2.56 2.07 2.13 2.35 2.42 1.76 1.94 1.78 1.91 1.21 2.64 2.17 2.23 2.33 2.17 SPA 0.85 0.81 1.07 0.69 0.95 0.56 0.48 0.52 0.57 0.53 0.95 1.07 0.99 0.87 0.71 0.63 0.45 0.66 0.58 0.55 S1P 6.77 6.57 6.89 4.46 6.58 13.81 13.06 16.23 16.00 19.77 8.11 6.75 7.18 9.85 3.17 13.48 14.05 14.16 16.39 12.82 C8-cer 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 C14-cer 0.17 0.16 0.31 0.13 0.25 0.02 0.00 0.02 0.06 0.04 0.33 0.14 0.28 0.12 0.06 0.03 0.03 0.04 0.03 0.02 C16-cer 1.40 1.11 1.98 1.17 1.63 0.93 0.70 0.81 1.54 1.33 1.54 0.98 1.30 1.27 0.84 0.92 0.78 1.02 0.77 0.92 C16-dh cer 0.15 0.18 0.35 0.15 0.00 0.15 0.00 0.12 0.00 0.12 0.23 0.20 0.17 0.16 0.15 0.12 0.00 0.12 0.14 0.12 C18:1-cer 0.35 0.13 0.35 0.19 0.36 0.00 0.00 0.00 0.00 0.25 0.28 0.28 0.20 0.29 0.16 0.00 0.00 0.07 0.00 0.08 C18:1-dh cer 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 C18-cer 128.08 123.77 166.72 114.65 160.63 109.24 93.23 82.11 109.77 112.30 175.62 122.60 133.60 152.42 104.56 96.24 102.47 108.97 96.68 102.63 C18-dh cer 7.22 9.61 6.84 9.17 12.94 2.54 2.06 1.40 1.27 2.45 11.13 9.99 9.82 10.45 7.50 1.82 2.40 1.89 2.07 2.28 C20-cer 35.58 41.13 41.78 32.81 44.02 30.34 19.67 21.01 24.06 30.08 52.84 34.99 41.13 46.19 24.87 25.42 25.79 30.61 26.24 28.81 C22-cer 42.67 51.85 47.67 38.91 57.70 47.30 28.76 37.43 49.15 44.43 67.31 55.58 50.00 60.32 23.12 32.70 34.01 33.64 35.20 31.57 C24:1-cer 88.46 84.75 85.17 83.37 99.29 93.01 73.91 80.51 100.24 93.94 98.58 92.57 98.41 103.67 65.97 79.67 78.44 77.33 86.26 77.62 C24-cer 19.56 22.87 18.96 16.99 24.59 14.78 8.75 10.71 14.48 13.69 27.85 25.59 22.93 20.73 12.02 9.27 10.89 10.32 10.92 9.97 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Sph SPA S1P C8-cer C14-cer C16-cer C16-dh cer C18:1-cer C18:1-dh cer C18-cer C18-dh cer C20-cer C22-cer C24:1-cer C24-cer METABOLITES_END #END