#METABOLOMICS WORKBENCH hormel101_20170711_145535 DATATRACK_ID:1118 STUDY_ID:ST000790 ANALYSIS_ID:AN001258 PROJECT_ID:PR000574 VERSION 1 CREATED_ON July 12, 2017, 2:37 pm #PROJECT PR:PROJECT_TITLE Mayo Pilot and Feasibility: Identifying metabolic adaptations characteristic of PR:PROJECT_TITLE multiple myeloma cells via mass spectrometry-based metabolite profiling PR:PROJECT_SUMMARY Mayo Pilot and Feasibility: Identifying metabolic adaptations characteristic of PR:PROJECT_SUMMARY multiple myeloma cells via mass spectrometry-based metabolite profiling PR:INSTITUTE Mayo Clinic PR:LAST_NAME Gonsalves PR:FIRST_NAME Wilson PR:ADDRESS 200 First St. SW, Rochester, Minnesota, 55905, USA PR:EMAIL gonsalves.wilson@mayo.edu PR:PHONE 507-266-0792 #STUDY ST:STUDY_TITLE Identifying metabolic adaptations characteristic of multiple myeloma cells via ST:STUDY_TITLE targeted sphingolipids concentrations from bone marrow and peripheral plasma ST:STUDY_SUMMARY Will be assessing the targeted sphingolipids concentrations of high risk versus ST:STUDY_SUMMARY low risk smoldering myeloma patients based on peripheral blood plasma and bone ST:STUDY_SUMMARY marrow plasma. ST:INSTITUTE Mayo Clinic ST:LAST_NAME Gonsalves ST:FIRST_NAME Wilson ST:ADDRESS 200 First St. SW, Rochester, Minnesota, 55905, USA ST:EMAIL gonsalves.wilson@mayo.edu ST:PHONE 507-266-0792 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - ms6273-1 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-2 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-3 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-4 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-5 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-6 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-7 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-8 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-9 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-10 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-11 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-12 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-13 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-14 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-15 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-16 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-17 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-18 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-19 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-20 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-21 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-22 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-23 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-24 grouping:Quick Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-25 grouping:Slow Progressors Sample type=plasma; type=peripheral SUBJECT_SAMPLE_FACTORS - ms6273-26 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-27 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-28 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-29 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-30 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-31 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-32 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-33 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-34 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-35 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-36 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-37 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-38 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-39 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-40 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-41 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-42 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-43 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-44 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-45 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-46 grouping:Slow Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-47 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-48 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-49 grouping:Quick Progressors Sample type=plasma; type=bone marrow SUBJECT_SAMPLE_FACTORS - ms6273-50 grouping:Quick Progressors Sample type=plasma; type=bone marrow #COLLECTION CO:COLLECTION_SUMMARY "In order to analyze the metabolites of clonal PCs (intracellular) and BM plasma CO:COLLECTION_SUMMARY (extracellular) separately, they have to be separated out from the BM samples. CO:COLLECTION_SUMMARY Thus, upon acquiring the BM samples from patients they will be placed in CO:COLLECTION_SUMMARY centrifuged at 2500 rpm for 10 minutes to separate out the plasma from the CO:COLLECTION_SUMMARY cellular fraction. The separated BM plasma is then stored in separate vials and CO:COLLECTION_SUMMARY snap frozen under liquid nitrogen for 20 seconds before storing at -80οC for CO:COLLECTION_SUMMARY further analysis. The leftover cellular component present as a pellet will be CO:COLLECTION_SUMMARY washed and reconstituted with an equal volume of RPMI 1640 medium. Erythrocytes CO:COLLECTION_SUMMARY are lysed using ammonium chloride lysing solution. After incubation on ice for 5 CO:COLLECTION_SUMMARY min, the cell suspension is diluted with RPMI medium. The cells are again CO:COLLECTION_SUMMARY pelleted by centrifugation and then suspended in RoboSep buffer (250ml PBS, 2% CO:COLLECTION_SUMMARY BSA, 1mM EDTA). The clonal CD138 positive PCs are purified using positive CO:COLLECTION_SUMMARY selection by mixing the cells with a CD138 positive selection cocktail and CO:COLLECTION_SUMMARY anti-CD138 magnetic-activated cell separation microbeads (ROBOSEP™ cell CO:COLLECTION_SUMMARY separation system, StemCell Technologies Inc) in the automated RoboSep cell CO:COLLECTION_SUMMARY separation system. The purified samples containing only CD138 positive cells are CO:COLLECTION_SUMMARY re‐suspended and then centrifuged to form a cell pellet. The goal will be to CO:COLLECTION_SUMMARY obtain at least 1-2 x 107 clonal PCs per sample. The cell pellet will be snap CO:COLLECTION_SUMMARY frozen under liquid nitrogen for 20 seconds before storing at -80οC for further CO:COLLECTION_SUMMARY analysis. Both the BM plasma samples as well as the clonal PCs pellet will be CO:COLLECTION_SUMMARY provided to the metabolomics core for sample preparation for LC-MS analysis. For CO:COLLECTION_SUMMARY Aim 2, we will be using stored BM samples from SMM patients that have already CO:COLLECTION_SUMMARY had their BM plasma and clonal PCs separated from each other and stored at CO:COLLECTION_SUMMARY -80οC. It is important to note that all these samples were collected and frozen CO:COLLECTION_SUMMARY in a timely manner. Furthermore consistency in sample collection, storage and CO:COLLECTION_SUMMARY processing is imperative for the optimal conduct of metabolomics-based CO:COLLECTION_SUMMARY experiments. This ensures that all samples being analyzed and compared with each CO:COLLECTION_SUMMARY other would have been manipulated similarly, limiting any handling biases. All CO:COLLECTION_SUMMARY patient samples in the Predolin Foundation Biobank were collected and stored by CO:COLLECTION_SUMMARY following a standardized operating procedure. BM samples were processed for BM CO:COLLECTION_SUMMARY plasma separation and clonal PCs enrichment but were then immediately snap CO:COLLECTION_SUMMARY frozen for storage at -80οC within approximately 3 hours of collection from the CO:COLLECTION_SUMMARY patient. All samples were collected in patients who have been fasting for at CO:COLLECTION_SUMMARY least 6 to 8 hours prior. To further ensure consistency in our analysis of this CO:COLLECTION_SUMMARY study, we will only use samples that have never been thawed since initial CO:COLLECTION_SUMMARY storage to preserve the stability of the metabolites originally present in the CO:COLLECTION_SUMMARY samples." #TREATMENT TR:TREATMENT_SUMMARY We will use matched BM plasma and purified clonal marrow PCs from the BM samples TR:TREATMENT_SUMMARY of SMM patients collected at the time of their diagnosis and stored within the TR:TREATMENT_SUMMARY Mayo Clinic Predolin Foundation Biobank. We will select BM samples from patients TR:TREATMENT_SUMMARY with SMM who progressed to MM within 2 years of their samples being collected TR:TREATMENT_SUMMARY and stored; this will constitute the high risk SMM group. We will also select BM TR:TREATMENT_SUMMARY samples from SMM patients who have not progressed to MM within at least 2 years TR:TREATMENT_SUMMARY of follow up of their samples being collected and stored; this will constitute TR:TREATMENT_SUMMARY the standard risk SMM group. The strength of this approach is that we will TR:TREATMENT_SUMMARY utilize stored SMM samples from one of the most comprehensively characterized TR:TREATMENT_SUMMARY monoclonal gammopathy biobanks available. Secondly, all samples will be obtained TR:TREATMENT_SUMMARY from collection dates at least 2 years prior in order to ensure adequate TR:TREATMENT_SUMMARY follow-up time to assess their current clinical status. This would avoid the TR:TREATMENT_SUMMARY need to prospectively collect samples from SMM patients and clinically follow TR:TREATMENT_SUMMARY them for a number of years before we are able to gauge who were clinically high TR:TREATMENT_SUMMARY or standard risk for progression. There are over 700 SMM patients who have had TR:TREATMENT_SUMMARY their BM samples collected and stored in the biobank from 1996 to 2013; more TR:TREATMENT_SUMMARY than half these patients have progressed to MM. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY sphingolipids concetrations #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:MS_COMMENTS - 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 uM MS_METABOLITE_DATA_START Samples ms6273-1 ms6273-2 ms6273-3 ms6273-4 ms6273-5 ms6273-6 ms6273-7 ms6273-8 ms6273-9 ms6273-10 ms6273-11 ms6273-12 ms6273-13 ms6273-14 ms6273-15 ms6273-16 ms6273-17 ms6273-18 ms6273-19 ms6273-20 ms6273-21 ms6273-22 ms6273-23 ms6273-24 ms6273-25 ms6273-26 ms6273-27 ms6273-28 ms6273-29 ms6273-30 ms6273-31 ms6273-32 ms6273-33 ms6273-34 ms6273-35 ms6273-36 ms6273-37 ms6273-38 ms6273-39 ms6273-40 ms6273-41 ms6273-42 ms6273-43 ms6273-44 ms6273-45 ms6273-46 ms6273-47 ms6273-48 ms6273-49 ms6273-50 Factors grouping:Slow Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Slow Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Quick Progressors grouping:Quick Progressors Sph 0.04 0.05 0.07 0.02 0.03 0.01 0.02 0.03 0.02 0.02 0.01 0.07 0.04 0.03 0.07 0.04 0.07 0.05 0.02 0.02 0.03 0.03 0.04 0.05 0.02 0.06 0.11 0.08 0.12 0.19 0.19 0.15 0.28 0.13 0.28 0.17 0.08 0.07 0.12 0.04 0.14 0.15 0.12 0.21 0.18 0.03 0.24 0.17 0.18 0.21 SPA 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 0.00 0.04 0.02 0.00 0.00 0.00 0.02 0.02 0.00 0.00 0.03 0.04 0.04 0.04 0.05 0.07 0.06 0.07 0.06 0.05 0.05 0.04 0.04 0.03 0.02 0.05 0.07 0.03 0.06 0.05 0.03 0.06 0.05 0.05 0.06 S1P 0.66 1.39 0.81 0.73 0.81 1.14 0.91 0.80 1.15 0.96 0.49 1.03 1.13 0.93 1.30 0.60 1.35 1.20 0.90 0.95 0.79 0.99 1.25 0.98 0.93 0.61 0.40 0.63 0.69 1.05 0.51 0.62 0.56 1.09 0.36 0.51 0.60 0.50 0.52 0.65 0.47 0.59 0.65 0.48 0.62 0.59 0.43 0.65 0.52 0.67 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 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 C14-cer 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 C16-cer 0.20 0.34 0.46 0.61 0.23 0.38 0.31 0.33 0.28 0.23 0.18 0.29 0.47 0.29 0.41 0.38 0.40 0.25 0.23 0.38 0.28 0.27 0.25 0.33 0.28 0.31 0.33 0.22 0.40 0.32 0.19 0.34 0.28 0.58 0.27 0.21 0.24 0.25 0.26 0.16 0.18 0.36 0.21 0.30 0.31 0.24 0.34 0.33 0.25 0.24 C18:1-cer 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.02 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 C18-cer 0.06 0.16 0.16 0.05 0.05 0.08 0.12 0.12 0.09 0.05 0.04 0.11 0.15 0.08 0.28 0.17 0.13 0.10 0.07 0.13 0.12 0.08 0.08 0.09 0.10 0.09 0.10 0.05 0.14 0.08 0.04 0.13 0.06 0.27 0.11 0.04 0.10 0.07 0.04 0.05 0.04 0.12 0.06 0.10 0.08 0.07 0.17 0.13 0.08 0.09 C20-cer 0.07 0.17 0.18 0.05 0.05 0.10 0.10 0.13 0.11 0.06 0.06 0.13 0.18 0.11 0.24 0.21 0.21 0.07 0.09 0.16 0.08 0.07 0.07 0.09 0.11 0.08 0.06 0.05 0.12 0.07 0.04 0.11 0.05 0.00 0.08 0.04 0.08 0.06 0.05 0.05 0.04 0.13 0.06 0.09 0.08 0.07 0.15 0.09 0.07 0.07 C22-cer 0.32 0.36 0.50 0.36 0.32 0.42 0.45 0.40 0.45 0.38 0.26 0.37 0.55 0.34 0.62 0.42 0.54 0.31 0.40 0.57 0.32 0.39 0.30 0.57 0.41 0.46 0.36 0.29 0.37 0.46 0.20 0.36 0.33 0.69 0.35 0.29 0.33 0.38 0.27 0.30 0.30 0.45 0.33 0.33 0.45 0.35 0.42 0.32 0.43 0.35 C24:1-cer 0.28 0.50 0.42 0.80 0.44 0.64 0.57 0.42 0.57 0.38 0.35 0.49 0.58 0.66 0.97 0.45 0.50 0.54 0.34 0.64 0.34 0.34 0.29 0.55 0.40 0.55 0.31 0.42 0.48 0.65 0.37 0.47 0.49 0.96 0.65 0.43 0.53 0.38 0.42 0.27 0.37 0.43 0.50 0.33 0.61 0.36 0.45 0.45 0.26 0.48 C24-cer 1.03 1.18 1.28 0.89 1.12 1.54 1.41 1.18 1.30 1.27 0.90 1.17 1.53 0.91 1.28 1.31 1.41 1.11 1.32 1.34 1.02 1.22 1.05 1.40 1.03 1.25 1.02 0.68 1.14 1.42 0.84 1.24 1.06 1.33 0.78 0.96 0.89 1.21 0.91 1.05 0.97 1.16 0.76 1.07 1.28 0.97 1.16 0.99 1.13 0.85 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Sph SPA S1P C8-cer C14-cer C16-cer C18:1-cer C18-cer C20-cer C22-cer C24:1-cer C24-cer METABOLITES_END #END