#METABOLOMICS WORKBENCH hormel101_20170810_150108_mwtab.txt DATATRACK_ID:1210 STUDY_ID:ST000859 ANALYSIS_ID:AN001384 PROJECT_ID:PR000575 VERSION 1 CREATED_ON August 11, 2017, 12:34 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 Galactosyl Sphingolipid Concentration in Kallikrein 6 Mice after SCI ST:STUDY_SUMMARY Targeted Galactosyl Sphingolipids in Kallikrein 6 Mice after SCI. The samples ST:STUDY_SUMMARY submitted are purified myelin preparations from the postnatal day 21, 60, or 90 ST:STUDY_SUMMARY mouse spinal cord (SC). There are 12 samples total in Project 3, n=3 for K6+/+ ST:STUDY_SUMMARY or K6-/- at 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 - ms6025-1 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-2 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-3 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-4 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-5 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-6 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-7 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-8 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-9 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-10 group:K6+/+ sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-11 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-12 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-13 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-14 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-15 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-16 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-17 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-18 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-19 group:K6-/- sex=Male; tissue=Tissue (SC); species=Mouse SUBJECT_SAMPLE_FACTORS - ms6025-20 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 "Myelin galactosyl sphinolipid concentrations from mouse spinal cord Lipids will SP:SAMPLEPREP_SUMMARY be quantified in myelin isolated in high yield and purity by subcellular SP:SAMPLEPREP_SUMMARY fractionation from the lumbosacral spinal cord. While there are no absolutely SP:SAMPLEPREP_SUMMARY ‘myelin-specific’ lipids, galactocerebroside is the most typical of myelin SP:SAMPLEPREP_SUMMARY in the adult nervous system being directly proportional to the amount of myelin. SP:SAMPLEPREP_SUMMARY Sulfatide is another galactolipid enriched in myelin. Together with cholesterol, SP:SAMPLEPREP_SUMMARY these form 78% of the total amount of lipid in the myelin membrane and each will SP:SAMPLEPREP_SUMMARY be quantified using LC/MS/MS. A highly sensitive assay for galactocerebroside SP:SAMPLEPREP_SUMMARY was recently established by the Mayo Metabolomics Core and can be implemented SP:SAMPLEPREP_SUMMARY immediately. The LC/MS/MS panel for free fatty acids, including the very long SP:SAMPLEPREP_SUMMARY chain fatty acids found in myelin is also routinely performed by the Core. SP:SAMPLEPREP_SUMMARY Cholesterol will be quantified using an NMR-based approach by the Mayo Dept. of SP:SAMPLEPREP_SUMMARY Laboratory Medicine Clinical Core. Additionally, we have a plan in place with SP:SAMPLEPREP_SUMMARY the 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 HILIC CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME AMT HALO HILIC 2.7, 4.6x150mm #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 ms6025-1 ms6025-2 ms6025-3 ms6025-4 ms6025-5 ms6025-6 ms6025-7 ms6025-8 ms6025-9 ms6025-10 ms6025-11 ms6025-12 ms6025-13 ms6025-14 ms6025-15 ms6025-16 ms6025-17 ms6025-18 ms6025-19 ms6025-20 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-/- Gal-Sph 0.29 not found not found not found not found 1.85 1.76 2.04 2.60 2.14 not found not found not found not found not found 1.73 1.59 1.21 1.40 2.36 Gal-C16-Cer 19 13 21 20 25 31 30 33 38 38 29 27 24 28 13 33 30 31 39 37 Gal-C18-Cer 877 617 864 787 957 1547 1266 1424 1539 1672 1039 1026 1040 1031 596 1505 1313 1256 1654 1613 Gal-C24:1-Cer 3330 2492 3296 3274 3798 6103 6179 6748 6923 6969 3895 3788 3814 4741 2562 6448 6497 6367 6975 6832 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Gal-Sph Gal-C16-Cer Gal-C18-Cer Gal-C24:1-Cer METABOLITES_END #END