#METABOLOMICS WORKBENCH hormel101_20160722_115828 DATATRACK_ID:685 STUDY_ID:ST000542 ANALYSIS_ID:AN000824 PROJECT_ID:PR000397 VERSION 1 CREATED_ON January 30, 2017, 9:53 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 Triple Quadrupole Mass Spectrometer to measure low abundance isotope enrichment ST:STUDY_TITLE in individual muscle proteins ST:STUDY_TYPE isotope encrichment and comparison of mass spectrometer platforms, timecourse 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 1A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject1 1A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject1 1B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject1 1B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject2 2A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject2 2A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject2 2B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject2 2B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject3 3A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject3 3A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject3 3B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject3 3B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject4 5A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject4 5A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject4 5B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject4 5B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject5 7A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject5 7A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject5 7B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject5 7B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject6 8A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject6 8A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject6 8B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject6 8B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject7 14A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject7 14A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject7 14B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject7 14B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject7 14C-180 group:C | time (mins):180 SUBJECT_SAMPLE_FACTORS subject7 14C-480 group:C | time (mins):480 SUBJECT_SAMPLE_FACTORS subject8 16A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject8 16A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject8 16B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject8 16B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject8 16C-180 group:C | time (mins):180 SUBJECT_SAMPLE_FACTORS subject8 16C-480 group:C | time (mins):480 SUBJECT_SAMPLE_FACTORS subject9 18A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject9 18A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject9 18B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject9 18B-480 group:B | time (mins):480 SUBJECT_SAMPLE_FACTORS subject10 19A-180 group:A | time (mins):180 SUBJECT_SAMPLE_FACTORS subject10 19A-480 group:A | time (mins):480 SUBJECT_SAMPLE_FACTORS subject10 19B-180 group:B | time (mins):180 SUBJECT_SAMPLE_FACTORS subject10 19B-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 SP:SAMPLEPREP_SUMMARY in 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 The methodologies used in this approach have been previously published [18]. CH:CHROMATOGRAPHY_SUMMARY Briefly, HPLC separation was performed using a Cohesive TX2 multiplexed HPLC CH:CHROMATOGRAPHY_SUMMARY system with a Supelco Ascentis Express C18 column (15cm × 2.1 mm, 2.7µm) at a CH:CHROMATOGRAPHY_SUMMARY flow rate of 0.2 mL min−1, at room temperature. Solvent A was 99% water CH:CHROMATOGRAPHY_SUMMARY (Fisher Scientific), 1% acetonitrile (Fisher Scientific) with 0.1% formic acid CH:CHROMATOGRAPHY_SUMMARY (SigmaAldrich), and solvent B was 99.9% acetonitrile and 0.1% formic acid. 10 CH:CHROMATOGRAPHY_SUMMARY µL of sample was injected and separated using a gradient of: 20-70% B from CH:CHROMATOGRAPHY_SUMMARY 0-5.75 min, 70-95% B from 5.75-6.75 min, 95% B from 6.75-8.75 min, 95-5% B from CH:CHROMATOGRAPHY_SUMMARY 8.75-9.75 min, and 5% B from 9.75-13.95 min. The column eluent was passed into a CH:CHROMATOGRAPHY_SUMMARY triple quadrupole tandem mass spectrometer (ABSciex API 5000) through the CH:CHROMATOGRAPHY_SUMMARY electrospray ionization source operating under the following conditions: ion CH:CHROMATOGRAPHY_SUMMARY spray voltage = 5000 V; temperature = 300 °C; curtain gas = 40 a.u.; gas 1 = 30 CH:CHROMATOGRAPHY_SUMMARY a.u.; gas 2 = 10 a.u.; declustering potential = 60 V; entrance potential = 10 V; CH:CHROMATOGRAPHY_SUMMARY collision energy = 35 V; collision cell exit potential = 13 V; collision gas = 6 CH:CHROMATOGRAPHY_SUMMARY a.u.. Analysis of [ring-13C6]-phenylalanine enrichment in gel spot extracts was CH:CHROMATOGRAPHY_SUMMARY performed under multiple reaction monitoring (MRM) conditions monitoring the m/z CH:CHROMATOGRAPHY_SUMMARY 228.2>127.0 and m/z 224.2>123.0 transitions, which corresponded to the m+6 and CH:CHROMATOGRAPHY_SUMMARY m+2 fragments, respectively. The m+6 to m+2 peak area ratios of the unknowns CH:CHROMATOGRAPHY_SUMMARY were compared to a [ring-13C6]-phenylalanine calibration curve prepared by CH:CHROMATOGRAPHY_SUMMARY mixing known amounts of [ring-13C6]-phenylalanine with a constant amount of CH:CHROMATOGRAPHY_SUMMARY natural abundance phenylalanine over a range of 0 – 0.132% enrichment, CH:CHROMATOGRAPHY_SUMMARY measured as mole percent enrichment (mpe). FSR calculations were based on the CH:CHROMATOGRAPHY_SUMMARY [ring-13C6]-phenylalanine enrichment results for the 2D-GE isolated muscle CH:CHROMATOGRAPHY_SUMMARY proteins as well as the enrichment in the corresponding tissue fluids, which CH:CHROMATOGRAPHY_SUMMARY were measured on the same platform. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Cohesive TX2 CH:COLUMN_NAME Supelco Ascentis Express C18 column #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex API 5000 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS mole percent enrichment MS_METABOLITE_DATA_START Samples 1A-180 1A-480 1B-180 1B-480 2A-180 2A-480 2B-180 2B-480 3A-180 3A-480 3B-180 3B-480 5A-180 5A-480 5B-180 5B-480 7A-180 7A-480 7B-180 7B-480 8A-180 8A-480 8B-180 8B-480 14A-180 14A-480 14B-180 14B-480 14C-180 14C-480 16A-180 16A-480 16B-180 16B-480 16C-180 16C-480 18A-180 18A-480 18B-180 18B-480 19A-180 19A-480 19B-180 19B-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 0.038 0.069 0.027 0.037 0.076 0.058 0.06 0.045 0.066 0.009 0.026 0.062 0.073 0.018 0.038 0.046 0.056 0.066 0.029 0.035 0.05 0.012 0.047 0.042 0.017 0.035 0.038 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID ATP Synthase 13c6 phe mpe METABOLITES_END #END