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MB Sample ID: SA028067
Local Sample ID: | R2A-180 |
Subject ID: | SU000565 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Human |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU000565 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Human |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
R2A-180 | SA028067 | FL006964 | A | group |
R2A-180 | SA028067 | FL006964 | 180 | time (mins) |
Collection:
Collection ID: | CO000559 |
Collection Summary: | Percutaneous needle biopsies of the vastus lateralis muscle were performed under local anesthesia at 180 min and 480 min into the infusion [3;21]. The studies were repeated 65-80 days following the first study. Explanation of study design factors: Study A is the first study period, Study B is the second study conducted after 655-80 days after the first, Time 180 is the first muscle biopsy within the given study period, Time 480 is the second muscle biopsy within the given study period. Study C is the third study conducted after the second for a few subjects. |
Sample Type: | Muscle |
Treatment:
Treatment ID: | TR000579 |
Treatment Summary: | We performed studies in healthy study participants in the Mayo Clinic Clinical Research Unit (CRU) after obtaining informed consent as a protocol approved by the Institutional Review Board. In ten healthy human study participants (age = 57.1 ± 20.24 yrs; M:F ratio = 1.5; and BMI = 26.80 ± 3.38 kg/mL), after overnight fast, we infused [ring-13C6]-phenylalanine at a rate of 1 mg/kg FFM/hr for eight hours following a priming dose to achieve an early isotope plateau [19;20]. |
Sample Preparation:
Sampleprep ID: | SP000572 |
Sampleprep Summary: | From the human muscle biopsies, 20-30 mg of quadriceps muscle was homogenized in urea buffer (9.8M urea, 4% CHAPS) and skeletal muscle mitochondria separated using a differential centrifugation [22]. Individual proteins were isolated from the mixture by performing large, high-resolution, 2D-GE [23]. Approximately 200 μg of each protein sample were dissolved in lysis buffer to a final volume of 450 μl. These samples were used to rehydrate 24-cm, pH 4–7 and 6–9, immobilized pH gradient (IPG) strips (Bio-Rad Laboratories, Hercules, CA) in a rehydration tray overnight. The rehydrated IPG strips were subjected to isoelectric focusing in a Protean IEF Cell (Bio-Rad) using a three-step protocol: i) the focusing was achieved with an initial step of 250 V for 15 min; ii) continued with a maximum of 10,000 V increased linearly from 250 V over 6 h; and iii) continued at 10,000 V for 6 h. The cell temperature was kept at 20°C with a maximum current of 50 μA per strip. The IPG strips were then equilibrated for the SDS-PAGE in a two-step equilibration using 5 mL of equilibration buffer per strip (6 M urea, 2% SDS, 0.375 M Tris·HCl, pH 8.8, and 20% glycerol) with 130 mM DTT in the first step and 135 mM iodoacetamide in the second step. The equilibration steps were done in an equilibration tray for 10 min each on a rotary shaker at room temperature. The second-dimension separation by subunit molecular weight was performed by vertical 12%, 24 × 20-cm dimension SDS-PAGE (Ettan DALT system; GE Healthcare Bio-Sciences, Piscataway, NJ). The IPG strips were mounted into the IPG well with molten agarose and then run at 75 V for 24 h or until the dye front reached the bottom of the gel. The protein gel spots were visualized by staining with Coomassie blue (GelCode Blue Stain Reagent; Pierce, Rockford, IL). Spots were excised from the gel, placed in glass vials, and washed several times with water. An additional 3 mL of HPLC water (Fisher Scientific) was added to each vial, and the gel spot samples were placed on a rocking shaker for 60 min. The proteins were then hydrolyzed at 120 °C for 18 h with 6 M HCl. The following day, the gel spot samples were centrifuged for 5 min at 3,000 rpm and 4 °C, after which 2 mL of water was added to each vial and vortexed. To prepare the AG-50x8 cation exchange column, the resin was rinsed with 4 mL of 4M ammonium hydroxide (NH4OH), followed by 4 rinses with 5 mL water. The column resin was regenerated with 4 mL of 4M HCl and rinsed with 5 mL of 0.1M HCl. The gel spot samples (approx. 2 mL) were transferred to the prepared AG-50 columns, the column was rinsed 4X with 4 mL of HPLC water, and amino acids were eluted into washed glass vials with three 1 mL washes of 4M NH4OH. The eluents were dried overnight in a speed-vac without heat. Amino acids were derivatized with 50 µL of 4M HCl in dry isobutanol at 85 °C for 45 min and dried under nitrogen. For LC-MS/MS analyses, 40 µL of 5% acetonitrile in water was added to each vial, vortexed and transferred to autosampler vials. |
Combined analysis:
Analysis ID | AN000825 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Thermo Dionex Ultimate 3000 |
Column | Zorbax Extended C18 |
MS Type | ESI |
MS instrument type | Orbitrap |
MS instrument name | Thermo Q Exactive Plus Orbitrap |
Ion Mode | POSITIVE |
Units | mole percent enrichment |
Chromatography:
Chromatography ID: | CH000589 |
Chromatography Summary: | UPLC separations were performed with a Dionex UltiMate 3000 system (ThermoFisher Scientific) and a Zorbax Extended C18 column (Agilent; 5 cm × 2.1 mm, 1.8 µm). Solvent A was 99% water (Fisher Scientific), 1% acetonitrile (Fisher Scientific) with 0.1% formic acid (SigmaAldrich), and solvent B was 99% acetonitrile, 1% water and 0.1% formic acid. The gradient was as follows: 0-6 min: 5-20% B; 6-10 min: 20-95% B; 10-12 min: 95% B; 12-13 min: 95-5% B, at a flow rate of 0.3 mL min−1. An injection volume of 5 µL was used. The UPLC was connected to the ion source through the diverter valve. The HESI ion source was operated with +3.5 kV spray voltage and probe heater temperature of 300 °C. Sheath, auxiliary and spare gas were 47.5, 11.25 and 1.0 (arb. units), respectively. The capillary temperature was maintained at 275 °C. |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | Zorbax Extended C18 |
Flow Gradient: | 0-6 min: 5-20% B; 6-10 min: 20-95% B; 10-12 min: 95% B; 12-13 min: 95-5% B |
Flow Rate: | 0.3ml/min |
Solvent A: | 99% water/1% acetonitrile; 0.1% formic acid |
Solvent B: | 99% acetonitrile/1% water; 0.1% formic acid |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS000726 |
Analysis ID: | AN000825 |
Instrument Name: | Thermo Q Exactive Plus Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
Ion Mode: | POSITIVE |