Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA028064

Local Sample ID:	R5A-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:

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.

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.