#METABOLOMICS WORKBENCH hormel101_20170809_154457_mwtab.txt DATATRACK_ID:1199 STUDY_ID:ST000848 ANALYSIS_ID:AN001372 PROJECT_ID:PR000575 VERSION 1 CREATED_ON August 10, 2017, 3:11 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 NEFA of Kallikrein 6 Signals through PAR1 and PAR2 to Enchance ST:STUDY_TITLE Recovery of Function after SCI ST:STUDY_SUMMARY Targeting mouse myelin NEFA of Kallikrein 6 Signals through PAR1 and PAR2 to ST:STUDY_SUMMARY Enchance Recovery of Function after SCI. The samples submitted are purified ST:STUDY_SUMMARY myelin preparations from the postnatal day 21, 60, or 90 mouse spinal cord (SC). ST:STUDY_SUMMARY There are 9 samples total in Project 1, n=3 for each genotype (control, PAR1 or ST:STUDY_SUMMARY PAR2) at 21 days. There are 9 samples total in Project 2, n=3 for each genotype ST:STUDY_SUMMARY (WT, PAR1-/- or PAR2-/-) at 60 days. There are 12 samples total in Project 3, ST:STUDY_SUMMARY 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 - ms5959-1 time pt:P21d | grouping:CTRL gender=Female; protein (ug/ul)=1.81 SUBJECT_SAMPLE_FACTORS - ms5959-2 time pt:P21d | grouping:CTRL gender=Female; protein (ug/ul)=1.57 SUBJECT_SAMPLE_FACTORS - ms5959-3 time pt:P21d | grouping:CTRL gender=Female; protein (ug/ul)=1.99 SUBJECT_SAMPLE_FACTORS - ms5959-4 time pt:P21d | grouping:PAR1 gender=Female; protein (ug/ul)=1.42 SUBJECT_SAMPLE_FACTORS - ms5959-5 time pt:P21d | grouping:PAR1 gender=Female; protein (ug/ul)=2.15 SUBJECT_SAMPLE_FACTORS - ms5959-6 time pt:P21d | grouping:PAR1 gender=Female; protein (ug/ul)=1.87 SUBJECT_SAMPLE_FACTORS - ms5959-7 time pt:P21d | grouping:PAR2 gender=Female; protein (ug/ul)=1.83 SUBJECT_SAMPLE_FACTORS - ms5959-8 time pt:P21d | grouping:PAR2 gender=Female; protein (ug/ul)=1.21 SUBJECT_SAMPLE_FACTORS - ms5959-9 time pt:P21d | grouping:PAR2 gender=Female; protein (ug/ul)=2.20 SUBJECT_SAMPLE_FACTORS - ms5959-10 time pt:P60d | grouping:Wt gender=Female; protein (ug/ul)=1.55 SUBJECT_SAMPLE_FACTORS - ms5959-11 time pt:P60d | grouping:Wt gender=Female; protein (ug/ul)=1.73 SUBJECT_SAMPLE_FACTORS - ms5959-12 time pt:P60d | grouping:Wt gender=Female; protein (ug/ul)=2.49 SUBJECT_SAMPLE_FACTORS - ms5959-13 time pt:P60d | grouping:PAR2-/- gender=Female; protein (ug/ul)=1.86 SUBJECT_SAMPLE_FACTORS - ms5959-14 time pt:P60d | grouping:PAR2-/- gender=Female; protein (ug/ul)=1.48 SUBJECT_SAMPLE_FACTORS - ms5959-15 time pt:P60d | grouping:PAR2-/- gender=Female; protein (ug/ul)=2.70 SUBJECT_SAMPLE_FACTORS - ms5959-16 time pt:P60d | grouping:PAR1-/- gender=Female; protein (ug/ul)=2.94 SUBJECT_SAMPLE_FACTORS - ms5959-17 time pt:P60d | grouping:PAR1-/- gender=Female; protein (ug/ul)=2.61 SUBJECT_SAMPLE_FACTORS - ms5959-18 time pt:P60d | grouping:PAR1-/- gender=Female; protein (ug/ul)=2.52 SUBJECT_SAMPLE_FACTORS - ms5959-19 time pt:P21d | grouping:K6+/+ gender=Male; protein (ug/ul)=2.61 SUBJECT_SAMPLE_FACTORS - ms5959-20 time pt:P21d | grouping:K6+/+ gender=Male; protein (ug/ul)=2.48 SUBJECT_SAMPLE_FACTORS - ms5959-21 time pt:P21d | grouping:K6+/+ gender=Male; protein (ug/ul)=1.93 SUBJECT_SAMPLE_FACTORS - ms5959-22 time pt:P21d | grouping:K6-/- gender=Male; protein (ug/ul)=1.72 SUBJECT_SAMPLE_FACTORS - ms5959-23 time pt:P21d | grouping:K6-/- gender=Male; protein (ug/ul)=2.16 SUBJECT_SAMPLE_FACTORS - ms5959-24 time pt:P21d | grouping:K6-/- gender=Male; protein (ug/ul)=2.47 SUBJECT_SAMPLE_FACTORS - ms5959-25 time pt:P90d | grouping:K6+/+ gender=Male; protein (ug/ul)=2.70 SUBJECT_SAMPLE_FACTORS - ms5959-26 time pt:P90d | grouping:K6+/+ gender=Male; protein (ug/ul)=2.57 SUBJECT_SAMPLE_FACTORS - ms5959-27 time pt:P90d | grouping:K6+/+ gender=Male; protein (ug/ul)=2.93 SUBJECT_SAMPLE_FACTORS - ms5959-28 time pt:P90d | grouping:K6-/- gender=Male; protein (ug/ul)=3.16 SUBJECT_SAMPLE_FACTORS - ms5959-29 time pt:P90d | grouping:K6-/- gender=Male; protein (ug/ul)=2.96 SUBJECT_SAMPLE_FACTORS - ms5959-30 time pt:P90d | grouping:K6-/- gender=Male; protein (ug/ul)=3.88 #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 "NEFA of mouse spinal cord Lipids will be quantified in myelin isolated in high SP:SAMPLEPREP_SUMMARY yield and purity by subcellular fractionation from the lumbosacral spinal cord. SP:SAMPLEPREP_SUMMARY While there are no absolutely ‘myelin-specific’ lipids, galactocerebroside SP:SAMPLEPREP_SUMMARY is the most typical of myelin in the adult nervous system being directly SP:SAMPLEPREP_SUMMARY proportional to the amount of myelin. Sulfatide is another galactolipid enriched SP:SAMPLEPREP_SUMMARY in myelin. Together with cholesterol, these form 78% of the total amount of SP:SAMPLEPREP_SUMMARY lipid in the myelin membrane and each will be quantified using LC/MS/MS. A SP:SAMPLEPREP_SUMMARY highly sensitive assay for galactocerebroside was recently established by the SP:SAMPLEPREP_SUMMARY Mayo Metabolomics Core and can be implemented immediately. The LC/MS/MS panel SP:SAMPLEPREP_SUMMARY for free fatty acids, including the very long chain fatty acids found in myelin SP:SAMPLEPREP_SUMMARY is also routinely performed by the Core. Cholesterol will be quantified using an SP:SAMPLEPREP_SUMMARY NMR-based approach by the Mayo Dept. of Laboratory Medicine Clinical Core. SP:SAMPLEPREP_SUMMARY Additionally, we have a plan in place with the Metabolomics Core to develop SP:SAMPLEPREP_SUMMARY LC/MS/MS assays for sulfatide and sphingomyelin during the Pilot proposal. SP:SAMPLEPREP_SUMMARY Having quantitative assays for each of these key myelin lipids will facilitate SP:SAMPLEPREP_SUMMARY our goal to comprehensively profile myelin lipid metabolism and will form SP:SAMPLEPREP_SUMMARY foundational assays for a future NIH grant focused on myelin metabolism." #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity BEH C18 (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/ug of protein MS_METABOLITE_DATA_START Samples ms5959-1 ms5959-2 ms5959-3 ms5959-4 ms5959-5 ms5959-6 ms5959-7 ms5959-8 ms5959-9 ms5959-10 ms5959-11 ms5959-12 ms5959-13 ms5959-14 ms5959-15 ms5959-16 ms5959-17 ms5959-18 ms5959-19 ms5959-20 ms5959-21 ms5959-22 ms5959-23 ms5959-24 ms5959-25 ms5959-26 ms5959-27 ms5959-28 ms5959-29 ms5959-30 Factors time pt:P21d | grouping:CTRL time pt:P21d | grouping:CTRL time pt:P21d | grouping:CTRL time pt:P21d | grouping:PAR1 time pt:P21d | grouping:PAR1 time pt:P21d | grouping:PAR1 time pt:P21d | grouping:PAR2 time pt:P21d | grouping:PAR2 time pt:P21d | grouping:PAR2 time pt:P60d | grouping:Wt time pt:P60d | grouping:Wt time pt:P60d | grouping:Wt time pt:P60d | grouping:PAR2-/- time pt:P60d | grouping:PAR2-/- time pt:P60d | grouping:PAR2-/- time pt:P60d | grouping:PAR1-/- time pt:P60d | grouping:PAR1-/- time pt:P60d | grouping:PAR1-/- time pt:P21d | grouping:K6+/+ time pt:P21d | grouping:K6+/+ time pt:P21d | grouping:K6+/+ time pt:P21d | grouping:K6-/- time pt:P21d | grouping:K6-/- time pt:P21d | grouping:K6-/- time pt:P90d | grouping:K6+/+ time pt:P90d | grouping:K6+/+ time pt:P90d | grouping:K6+/+ time pt:P90d | grouping:K6-/- time pt:P90d | grouping:K6-/- time pt:P90d | grouping:K6-/- EPA 0.17 0.17 0.18 0.23 0.20 0.18 0.16 0.13 0.16 0.13 0.11 0.07 0.07 0.08 0.06 0.08 0.09 0.11 0.10 0.13 0.12 0.16 0.15 0.17 0.06 0.07 0.06 0.05 0.06 0.08 linolenic 0.02 0.03 0.03 0.03 0.19 0.02 0.01 0.02 0.03 0.03 0.09 0.02 0.02 0.03 0.01 0.03 0.02 0.03 0.01 0.02 0.02 0.03 0.04 0.04 0.02 0.03 0.02 0.02 0.03 0.03 DHA 0.63 0.63 0.73 0.84 0.71 0.56 0.53 0.70 0.75 0.53 0.76 0.51 0.50 0.61 0.64 0.44 0.59 0.61 0.71 0.67 0.79 0.96 0.80 0.94 0.32 0.43 0.38 0.38 0.47 0.53 myristic 0.25 0.18 0.21 0.31 0.51 0.17 0.20 0.31 0.27 0.29 0.33 0.11 0.12 0.13 0.12 0.12 0.15 0.16 0.14 0.20 0.25 0.18 0.15 0.29 0.14 0.33 0.07 0.08 0.10 0.16 palmitoleic 0.24 0.23 0.27 0.33 0.29 0.24 0.19 0.24 0.26 0.16 0.22 0.15 0.14 0.17 0.14 0.11 0.15 0.12 0.19 0.22 0.27 0.31 0.25 0.34 0.07 0.12 0.09 0.07 0.08 0.19 arachidonic 5.16 4.05 4.03 6.46 4.39 5.68 3.54 3.91 4.57 3.46 3.65 3.02 2.56 2.83 2.58 3.14 3.85 3.15 3.34 3.82 4.16 4.87 4.08 5.96 2.06 2.13 2.15 1.54 1.53 2.56 palmitelaidic 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 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 linoleic 0.75 0.76 0.80 1.06 0.79 0.85 0.68 0.86 0.87 0.63 1.31 0.65 0.56 0.60 0.50 0.60 0.62 0.58 0.68 0.79 0.82 0.94 0.78 1.06 0.45 0.66 0.52 0.49 0.54 0.57 palmitic 4.75 5.18 4.42 6.68 6.03 5.22 4.35 5.81 4.65 7.06 7.22 5.19 5.48 6.77 5.23 3.87 4.35 3.58 4.11 4.59 5.72 6.12 4.53 7.33 3.21 4.14 3.95 3.10 3.31 4.03 oleic 7.14 7.16 6.67 9.91 6.27 8.21 6.10 8.13 6.57 7.81 8.26 6.98 6.71 8.16 6.17 5.34 6.43 5.21 5.66 6.05 7.14 8.88 7.07 8.81 4.04 4.53 4.65 3.48 3.60 5.47 elaidic 0.00 0.00 0.00 0.00 0.03 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 0.03 0.01 0.07 0.00 0.00 0.00 0.04 stearic 4.88 5.53 3.82 6.79 6.48 6.16 5.22 5.70 4.22 6.89 5.94 4.46 4.91 5.78 4.14 3.63 3.61 2.25 3.99 4.39 6.04 6.17 4.28 7.66 3.12 3.75 4.02 2.83 2.77 3.41 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name EPA linolenic DHA myristic palmitoleic arachidonic palmitelaidic linoleic palmitic oleic elaidic stearic METABOLITES_END #END