#METABOLOMICS WORKBENCH hormel101_20170810_105321_mwtab.txt DATATRACK_ID:1203 STUDY_ID:ST000852 ANALYSIS_ID:AN001376 PROJECT_ID:PR000575 VERSION 1 CREATED_ON August 10, 2017, 3:39 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 Galactosyl Sphingolipids of Kallikrein 6 Signals through PAR1 ST:STUDY_TITLE and PAR2 to Enchance Recovery of Function after SCI ST:STUDY_SUMMARY Targeting mouse myelin Galactosyl Shingolipids of Kallikrein 6 Signals through ST:STUDY_SUMMARY PAR1 and PAR2 to Enchance Recovery of Function after SCI. The samples submitted ST:STUDY_SUMMARY are purified myelin preparations from the postnatal day 21, 60, or 90 mouse ST:STUDY_SUMMARY spinal 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 - ms5963-1 Grouping:CTRL Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-2 Grouping:CTRL Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-3 Grouping:CTRL Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-4 Grouping:PAR1 Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-5 Grouping:PAR1 Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-6 Grouping:PAR1 Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-7 Grouping:PAR2 Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-8 Grouping:PAR2 Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-9 Grouping:PAR2 Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-10 Grouping:Wt Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-11 Grouping:Wt Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-12 Grouping:Wt Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-13 Grouping:PAR2-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-14 Grouping:PAR2-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-15 Grouping:PAR2-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-16 Grouping:PAR1-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-17 Grouping:PAR1-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-18 Grouping:PAR1-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-19 Grouping:K6+/+ Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-20 Grouping:K6+/+ Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-21 Grouping:K6+/+ Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-22 Grouping:K6-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-23 Grouping:K6-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-24 Grouping:K6-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-25 Grouping:K6+/+ Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-26 Grouping:K6+/+ Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-27 Grouping:K6+/+ Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-28 Grouping:K6-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-29 Grouping:K6-/- Species=Mouse SUBJECT_SAMPLE_FACTORS - ms5963-30 Grouping:K6-/- Species=Mouse #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 "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 ms5963-1 ms5963-2 ms5963-3 ms5963-4 ms5963-5 ms5963-6 ms5963-7 ms5963-9 ms5963-10 ms5963-11 ms5963-12 ms5963-13 ms5963-14 ms5963-15 ms5963-16 ms5963-17 ms5963-18 ms5963-19 ms5963-20 ms5963-21 ms5963-22 ms5963-23 ms5963-24 ms5963-25 ms5963-26 ms5963-27 ms5963-28 ms5963-29 ms5963-30 Factors Grouping:CTRL Grouping:CTRL Grouping:CTRL Grouping:PAR1 Grouping:PAR1 Grouping:PAR1 Grouping:PAR2 Grouping:PAR2 Grouping:Wt Grouping:Wt Grouping:Wt Grouping:PAR2-/- Grouping:PAR2-/- Grouping:PAR2-/- Grouping:PAR1-/- Grouping:PAR1-/- Grouping:PAR1-/- Grouping:K6+/+ Grouping:K6+/+ Grouping:K6+/+ Grouping:K6-/- Grouping:K6-/- Grouping:K6-/- Grouping:K6+/+ Grouping:K6+/+ Grouping:K6+/+ Grouping:K6-/- Grouping:K6-/- Grouping:K6-/- Gal-C12-Cer 13.1 12.2 11.2 5.9 10.1 10.3 10.6 10.7 11.2 14.5 12.6 10.5 12.0 10.3 11.4 14.5 11.6 8.6 10.2 10.7 11.1 10.4 10.1 8.5 7.9 9.3 9.3 9.4 9.2 Gal-C16-Cer 23.7 25.3 25.9 10.7 20.7 22.7 17.5 21.1 20.1 23.2 21.3 17.4 24.5 19.7 19.0 21.1 22.2 16.0 18.2 18.9 22.3 23.0 20.6 13.4 14.6 14.1 15.0 16.3 15.5 Gal-C18-Cer 786 918 954 471 755 852 805 850 944 882 893 899 1103 912 795 883 959 711 787 783 888 874 830 680 777 741 720 800 746 Gal-C24:1-Cer 4594 4494 4508 2578 3609 3939 3837 3991 5177 5830 5425 4831 5623 5087 5036 5152 5487 3490 3700 3862 4204 3986 3883 4481 4929 5025 5077 5118 4920 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Gal-C12-Cer Gal-C16-Cer Gal-C18-Cer Gal-C24:1-Cer METABOLITES_END #END