#METABOLOMICS WORKBENCH hormel101_20160722_120948_mwtab.txt DATATRACK_ID:686 STUDY_ID:ST000543 ANALYSIS_ID:AN000825 PROJECT_ID:PR000397 VERSION 1 CREATED_ON January 30, 2017, 9:54 am #PROJECT PR:PROJECT_TITLE Application of high-resolution mass spectrometry to measure low abundance PR:PROJECT_TITLE isotope enrichment in individual muscle proteins PR:PROJECT_TYPE MS targeted analysis of [ring-13C6]-phenylalanine PR:PROJECT_SUMMARY Comparison of mass spectrometer methods to the analysis of PR:PROJECT_SUMMARY [ring-13C6]-phenylalanine enrichment in individual muscle proteins isolated with PR:PROJECT_SUMMARY 2D-GE PR:INSTITUTE Mayo Clinic PR:DEPARTMENT Endocrinology PR:LABORATORY Mayo Clinic Metabolomics Resource Core PR:LAST_NAME Nair PR:FIRST_NAME Sreekumaran PR:ADDRESS 200 First Street SW, Rochester, MN 55905 PR:EMAIL Nair.K@mayo.edu PR:PHONE 507-285-2415 #STUDY ST:STUDY_TITLE High Resolution orbittrap Mass Spectrometer to measure low abundance isotope ST:STUDY_TITLE enrichment in individual muscle proteins ST:STUDY_TYPE isotope encrichment and comparison of mass spectrometer platforms ST:STUDY_SUMMARY Stable isotope-labeled amino acids have long been used to measure the fractional ST:STUDY_SUMMARY synthesis rate of proteins, although the mass spectrometry platforms used for ST:STUDY_SUMMARY such analyses have changed throughout the years. More recently, tandem mass ST:STUDY_SUMMARY spectrometers such as triple quadrupoles have been accepted as the standard ST:STUDY_SUMMARY platform for enrichment measurement due to their sensitivity and the enhanced ST:STUDY_SUMMARY specificity offered by multiple reaction monitoring (MRM) experiments. The limit ST:STUDY_SUMMARY in the utility of such platforms for enrichment analysis occurs when measuring ST:STUDY_SUMMARY very low levels of enrichment from small amounts of sample, particularly ST:STUDY_SUMMARY proteins isolated from two-dimensional gel electrophoresis (2D-GE), where ST:STUDY_SUMMARY interference from contaminant ions impact the sensitivity of the measurement. We ST:STUDY_SUMMARY therefore applied a high resolution orbitrap mass spectrometer to the analysis ST:STUDY_SUMMARY of [ring-13C6]-phenylalanine enrichment in individual muscle proteins isolated ST:STUDY_SUMMARY with 2D-GE. Comparison of samples analyzed on both platforms revealed that the ST:STUDY_SUMMARY high resolution MS has significantly improved sensitivity relative to the triple ST:STUDY_SUMMARY quadrupole MS at very low-level enrichments due to its ability to resolve ST:STUDY_SUMMARY interferences in the m/z dimension. ST:INSTITUTE Mayo Clinic ST:DEPARTMENT Endocrinology ST:LABORATORY Mayo Clinic Metabolomics Resource Core ST:LAST_NAME Nair ST:FIRST_NAME Sreekumaran ST:ADDRESS 200 First Street SW, Rochester, MN 55905 ST:EMAIL Nair.K@mayo.edu ST:PHONE 507-285-2415 #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 subject1 R17A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject1 R17A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject1 R17B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject1 R17B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject2 R16A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject2 R16A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject2 R16B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject2 R16B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject3 R15A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject3 R15A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject3 R15B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject3 R15B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject4 R12A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject4 R12A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject4 R12B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject4 R12B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject5 R13A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject5 R13A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject5 R13B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject5 R13B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject6 R10A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject6 R10A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject6 R10B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject6 R10B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject7 R2A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject7 R2A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject7 R2B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject7 R2B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject7 R2C-180 group:C | time (mins):180 SUBJECT_SAMPLE_FACTORS subject7 R2C-480 group:C | time (mins):480 SUBJECT_SAMPLE_FACTORS subject8 R3A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject8 R3A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject8 R3B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject8 R3B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject8 R3C-180 group:C | time (mins):180 SUBJECT_SAMPLE_FACTORS subject8 R3C-480 group:C | time (mins):480 SUBJECT_SAMPLE_FACTORS subject9 R5A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject9 R5A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject9 R5B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject9 R5B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject10 R6A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject10 R6A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject10 R6B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject10 R6B-480 group:B | time (mins):480 #COLLECTION CO:COLLECTION_SUMMARY Percutaneous needle biopsies of the vastus lateralis muscle were performed under CO:COLLECTION_SUMMARY local anesthesia at 180 min and 480 min into the infusion [3;21]. The studies CO:COLLECTION_SUMMARY were repeated 65-80 days following the first study. Explanation of study design CO:COLLECTION_SUMMARY factors: Study A is the first study period, Study B is the second study CO:COLLECTION_SUMMARY conducted after 655-80 days after the first, Time 180 is the first muscle biopsy CO:COLLECTION_SUMMARY within the given study period, Time 480 is the second muscle biopsy within the CO:COLLECTION_SUMMARY given study period. Study C is the third study conducted after the second for a CO:COLLECTION_SUMMARY few subjects. #TREATMENT TR:TREATMENT_SUMMARY We performed studies in healthy study participants in the Mayo Clinic Clinical TR:TREATMENT_SUMMARY Research Unit (CRU) after obtaining informed consent as a protocol approved by TR:TREATMENT_SUMMARY the Institutional Review Board. In ten healthy human study participants (age = TR:TREATMENT_SUMMARY 57.1 ± 20.24 yrs; M:F ratio = 1.5; and BMI = 26.80 ± 3.38 kg/mL), after TR:TREATMENT_SUMMARY overnight fast, we infused [ring-13C6]-phenylalanine at a rate of 1 mg/kg FFM/hr TR:TREATMENT_SUMMARY for eight hours following a priming dose to achieve an early isotope plateau TR:TREATMENT_SUMMARY [19;20]. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY From the human muscle biopsies, 20-30 mg of quadriceps muscle was homogenized in SP:SAMPLEPREP_SUMMARY urea buffer (9.8M urea, 4% CHAPS) and skeletal muscle mitochondria separated SP:SAMPLEPREP_SUMMARY using a differential centrifugation [22]. Individual proteins were isolated from SP:SAMPLEPREP_SUMMARY the mixture by performing large, high-resolution, 2D-GE [23]. Approximately 200 SP:SAMPLEPREP_SUMMARY μg of each protein sample were dissolved in lysis buffer to a final volume of SP:SAMPLEPREP_SUMMARY 450 μl. These samples were used to rehydrate 24-cm, pH 4–7 and 6–9, SP:SAMPLEPREP_SUMMARY immobilized pH gradient (IPG) strips (Bio-Rad Laboratories, Hercules, CA) in a SP:SAMPLEPREP_SUMMARY rehydration tray overnight. The rehydrated IPG strips were subjected to SP:SAMPLEPREP_SUMMARY isoelectric focusing in a Protean IEF Cell (Bio-Rad) using a three-step SP:SAMPLEPREP_SUMMARY protocol: i) the focusing was achieved with an initial step of 250 V for 15 min; SP:SAMPLEPREP_SUMMARY ii) continued with a maximum of 10,000 V increased linearly from 250 V over 6 h; SP:SAMPLEPREP_SUMMARY and iii) continued at 10,000 V for 6 h. The cell temperature was kept at 20°C SP:SAMPLEPREP_SUMMARY with a maximum current of 50 μA per strip. The IPG strips were then SP:SAMPLEPREP_SUMMARY equilibrated for the SDS-PAGE in a two-step equilibration using 5 mL of SP:SAMPLEPREP_SUMMARY equilibration buffer per strip (6 M urea, 2% SDS, 0.375 M Tris·HCl, pH 8.8, and SP:SAMPLEPREP_SUMMARY 20% glycerol) with 130 mM DTT in the first step and 135 mM iodoacetamide in the SP:SAMPLEPREP_SUMMARY second step. The equilibration steps were done in an equilibration tray for 10 SP:SAMPLEPREP_SUMMARY min each on a rotary shaker at room temperature. The second-dimension separation SP:SAMPLEPREP_SUMMARY by subunit molecular weight was performed by vertical 12%, 24 × 20-cm dimension SP:SAMPLEPREP_SUMMARY SDS-PAGE (Ettan DALT system; GE Healthcare Bio-Sciences, Piscataway, NJ). The SP:SAMPLEPREP_SUMMARY IPG strips were mounted into the IPG well with molten agarose and then run at 75 SP:SAMPLEPREP_SUMMARY V for 24 h or until the dye front reached the bottom of the gel. The protein gel SP:SAMPLEPREP_SUMMARY spots were visualized by staining with Coomassie blue (GelCode Blue Stain SP:SAMPLEPREP_SUMMARY Reagent; Pierce, Rockford, IL). Spots were excised from the gel, placed in glass SP:SAMPLEPREP_SUMMARY vials, and washed several times with water. An additional 3 mL of HPLC water SP:SAMPLEPREP_SUMMARY (Fisher Scientific) was added to each vial, and the gel spot samples were placed SP:SAMPLEPREP_SUMMARY on a rocking shaker for 60 min. The proteins were then hydrolyzed at 120 °C for SP:SAMPLEPREP_SUMMARY 18 h with 6 M HCl. The following day, the gel spot samples were centrifuged for SP:SAMPLEPREP_SUMMARY 5 min at 3,000 rpm and 4 °C, after which 2 mL of water was added to each vial SP:SAMPLEPREP_SUMMARY and vortexed. To prepare the AG-50x8 cation exchange column, the resin was SP:SAMPLEPREP_SUMMARY rinsed with 4 mL of 4M ammonium hydroxide (NH4OH), followed by 4 rinses with 5 SP:SAMPLEPREP_SUMMARY mL water. The column resin was regenerated with 4 mL of 4M HCl and rinsed with 5 SP:SAMPLEPREP_SUMMARY mL of 0.1M HCl. The gel spot samples (approx. 2 mL) were transferred to the SP:SAMPLEPREP_SUMMARY prepared AG-50 columns, the column was rinsed 4X with 4 mL of HPLC water, and SP:SAMPLEPREP_SUMMARY amino acids were eluted into washed glass vials with three 1 mL washes of 4M SP:SAMPLEPREP_SUMMARY NH4OH. The eluents were dried overnight in a speed-vac without heat. Amino acids SP:SAMPLEPREP_SUMMARY were derivatized with 50 µL of 4M HCl in dry isobutanol at 85 °C for 45 min SP:SAMPLEPREP_SUMMARY and dried under nitrogen. For LC-MS/MS analyses, 40 µL of 5% acetonitrile in SP:SAMPLEPREP_SUMMARY water was added to each vial, vortexed and transferred to autosampler vials. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY UPLC separations were performed with a Dionex UltiMate 3000 system (ThermoFisher CH:CHROMATOGRAPHY_SUMMARY Scientific) and a Zorbax Extended C18 column (Agilent; 5 cm × 2.1 mm, 1.8 µm). CH:CHROMATOGRAPHY_SUMMARY Solvent A was 99% water (Fisher Scientific), 1% acetonitrile (Fisher Scientific) CH:CHROMATOGRAPHY_SUMMARY with 0.1% formic acid (SigmaAldrich), and solvent B was 99% acetonitrile, 1% CH:CHROMATOGRAPHY_SUMMARY water and 0.1% formic acid. The gradient was as follows: 0-6 min: 5-20% B; 6-10 CH:CHROMATOGRAPHY_SUMMARY min: 20-95% B; 10-12 min: 95% B; 12-13 min: 95-5% B, at a flow rate of 0.3 mL CH:CHROMATOGRAPHY_SUMMARY min−1. An injection volume of 5 µL was used. The UPLC was connected to the CH:CHROMATOGRAPHY_SUMMARY ion source through the diverter valve. The HESI ion source was operated with CH:CHROMATOGRAPHY_SUMMARY +3.5 kV spray voltage and probe heater temperature of 300 °C. Sheath, auxiliary CH:CHROMATOGRAPHY_SUMMARY and spare gas were 47.5, 11.25 and 1.0 (arb. units), respectively. The capillary CH:CHROMATOGRAPHY_SUMMARY temperature was maintained at 275 °C. CH:CHROMATOGRAPHY_TYPE Reversed phase MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap CH:COLUMN_NAME Zorbax Extended C18 column #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS mole percent enrichment MS_METABOLITE_DATA_START Samples R17A-180 R17A-480 R17B-180 R17B-480 R16A-180 R16A-480 R16B-180 R16B-480 R15A-180 R15A-480 R15B-180 R15B-480 R12A-180 R12A-480 R12B-180 R12B-480 R13A-180 R13A-480 R13B-180 R13B-480 R10A-180 R10A-480 R10B-180 R10B-480 R2A-180 R2A-480 R2B-180 R2B-480 R2C-180 R2C-480 R3A-180 R3A-480 R3B-180 R3B-480 R3C-180 R3C-480 R5A-180 R5A-480 R5B-180 R5B-480 R6A-180 R6A-480 R6B-180 R6B-480 Factors group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 group:C | time (mins):180 group:C | time (mins):480 group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 group:C | time (mins):180 group:C | time (mins):480 group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 group:A | time (mins):180 group:A | time (mins):480 group:B | time (mins):180 group:B | time (mins):480 ATP Synthase 13c6 phe mpe 0.0096 0.0274 0.0075 0.014 0.0214 0.0456 0.0623 0.0066 0.03 0.032 0.0691 0.0079 0.0095 0.0305 0.0526 0.0122 0.0165 0.0409 0.0307 0.0035 0.0093 0.0193 0.0401 0.0115 0.0598 0.0352 0.0444 0.0646 0.0754 0.0105 0.0313 0.046 0.0417 0.0741 0.1056 0.0104 0.0356 0.0412 0.0407 0.0732 0.0172 0.0369 0.0559 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name ATP Synthase 13c6 phe mpe METABOLITES_END #END