Summary of Study ST000542
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000397. The data can be accessed directly via it's Project DOI: 10.21228/M8R013 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000542 |
| Study Title | Triple Quadrupole Mass Spectrometer to measure low abundance isotope enrichment in individual muscle proteins |
| Study Type | isotope encrichment and comparison of mass spectrometer platforms, timecourse |
| Study Summary | Stable isotope-labeled amino acids have long been used to measure the fractional synthesis rate of proteins, although the mass spectrometry platforms used for such analyses have changed throughout the years. More recently, tandem mass spectrometers such as triple quadrupoles have been accepted as the standard platform for enrichment measurement due to their sensitivity and the enhanced specificity offered by multiple reaction monitoring (MRM) experiments. The limit in the utility of such platforms for enrichment analysis occurs when measuring very low levels of enrichment from small amounts of sample, particularly proteins isolated from two-dimensional gel electrophoresis (2D-GE), where interference from contaminant ions impact the sensitivity of the measurement. We therefore applied a high resolution orbitrap mass spectrometer to the analysis of [ring-13C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE. Comparison of samples analyzed on both platforms revealed that the high resolution MS has significantly improved sensitivity relative to the triple quadrupole MS at very low-level enrichments due to its ability to resolve interferences in the m/z dimension. |
| Institute | Mayo Clinic |
| Department | Endocrinology |
| Laboratory | Mayo Clinic Metabolomics Resource Core |
| Last Name | Nair |
| First Name | Sreekumaran |
| Address | 200 First Street SW, Rochester, MN 55905 |
| Nair.K@mayo.edu | |
| Phone | 507-285-2415 |
| Submit Date | 2016-04-11 |
| Analysis Type Detail | LC-MS |
| Release Date | 2017-07-10 |
| Release Version | 1 |
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Project:
| Project ID: | PR000397 |
| Project DOI: | doi: 10.21228/M8R013 |
| Project Title: | Application of high-resolution mass spectrometry to measure low abundance isotope enrichment in individual muscle proteins |
| Project Type: | MS targeted analysis of [ring-13C6]-phenylalanine |
| Project Summary: | Comparison of mass spectrometer methods to the analysis of [ring-13C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE |
| Institute: | Mayo Clinic |
| Department: | Endocrinology |
| Laboratory: | Mayo Clinic Metabolomics Resource Core |
| Last Name: | Nair |
| First Name: | Sreekumaran |
| Address: | 200 First Street SW, Rochester, MN 55905 |
| Email: | Nair.K@mayo.edu |
| Phone: | 507-285-2415 |
Subject:
| Subject ID: | SU000564 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Human |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | group | time (mins) |
|---|---|---|---|
| SA028019 | 7A-180 | A | 180 |
| SA028020 | 18A-180 | A | 180 |
| SA028021 | 16A-180 | A | 180 |
| SA028022 | 1A-180 | A | 180 |
| SA028023 | 14A-180 | A | 180 |
| SA028024 | 3A-180 | A | 180 |
| SA028025 | 8A-180 | A | 180 |
| SA028026 | 5A-180 | A | 180 |
| SA028027 | 19A-180 | A | 180 |
| SA028028 | 2A-180 | A | 180 |
| SA028029 | 16A-480 | A | 480 |
| SA028030 | 1A-480 | A | 480 |
| SA028031 | 2A-480 | A | 480 |
| SA028032 | 7A-480 | A | 480 |
| SA028033 | 8A-480 | A | 480 |
| SA028034 | 19A-480 | A | 480 |
| SA028035 | 18A-480 | A | 480 |
| SA028036 | 3A-480 | A | 480 |
| SA028037 | 14A-480 | A | 480 |
| SA028038 | 5A-480 | A | 480 |
| SA028039 | 18B-180 | B | 180 |
| SA028040 | 14B-180 | B | 180 |
| SA028041 | 19B-180 | B | 180 |
| SA028042 | 16B-180 | B | 180 |
| SA028043 | 8B-180 | B | 180 |
| SA028044 | 2B-180 | B | 180 |
| SA028045 | 5B-180 | B | 180 |
| SA028046 | 7B-180 | B | 180 |
| SA028047 | 3B-180 | B | 180 |
| SA028048 | 1B-180 | B | 180 |
| SA028049 | 2B-480 | B | 480 |
| SA028050 | 18B-480 | B | 480 |
| SA028051 | 3B-480 | B | 480 |
| SA028052 | 8B-480 | B | 480 |
| SA028053 | 16B-480 | B | 480 |
| SA028054 | 14B-480 | B | 480 |
| SA028055 | 1B-480 | B | 480 |
| SA028056 | 7B-480 | B | 480 |
| SA028057 | 5B-480 | B | 480 |
| SA028058 | 19B-480 | B | 480 |
| SA028059 | 16C-180 | C | 180 |
| SA028060 | 14C-180 | C | 180 |
| SA028061 | 16C-480 | C | 480 |
| SA028062 | 14C-480 | C | 480 |
| Showing results 1 to 44 of 44 |
Collection:
| Collection ID: | CO000558 |
| 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: | TR000578 |
| 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: | SP000571 |
| 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 | AN000824 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Cohesive TX2 |
| Column | Supelco Ascentis Express C18 |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex API 5000 QQQ |
| Ion Mode | POSITIVE |
| Units | mole percent enrichment |
Chromatography:
| Chromatography ID: | CH000588 |
| Chromatography Summary: | The methodologies used in this approach have been previously published [18]. Briefly, HPLC separation was performed using a Cohesive TX2 multiplexed HPLC system with a Supelco Ascentis Express C18 column (15cm × 2.1 mm, 2.7µm) at a flow rate of 0.2 mL min−1, at room temperature. Solvent A was 99% water (Fisher Scientific), 1% acetonitrile (Fisher Scientific) with 0.1% formic acid (SigmaAldrich), and solvent B was 99.9% acetonitrile and 0.1% formic acid. 10 µL of sample was injected and separated using a gradient of: 20-70% B from 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 8.75-9.75 min, and 5% B from 9.75-13.95 min. The column eluent was passed into a triple quadrupole tandem mass spectrometer (ABSciex API 5000) through the electrospray ionization source operating under the following conditions: ion spray voltage = 5000 V; temperature = 300 °C; curtain gas = 40 a.u.; gas 1 = 30 a.u.; gas 2 = 10 a.u.; declustering potential = 60 V; entrance potential = 10 V; collision energy = 35 V; collision cell exit potential = 13 V; collision gas = 6 a.u.. Analysis of [ring-13C6]-phenylalanine enrichment in gel spot extracts was performed under multiple reaction monitoring (MRM) conditions monitoring the m/z 228.2>127.0 and m/z 224.2>123.0 transitions, which corresponded to the m+6 and m+2 fragments, respectively. The m+6 to m+2 peak area ratios of the unknowns were compared to a [ring-13C6]-phenylalanine calibration curve prepared by mixing known amounts of [ring-13C6]-phenylalanine with a constant amount of natural abundance phenylalanine over a range of 0 – 0.132% enrichment, measured as mole percent enrichment (mpe). FSR calculations were based on the [ring-13C6]-phenylalanine enrichment results for the 2D-GE isolated muscle proteins as well as the enrichment in the corresponding tissue fluids, which were measured on the same platform. |
| Instrument Name: | Cohesive TX2 |
| Column Name: | Supelco Ascentis Express C18 |
| Column Temperature: | RT |
| Flow Gradient: | 20-70% B from 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 8.75-9.75 min, and 5% B from 9.75-13.95 min. |
| Flow Rate: | 0.2ml/min |
| Solvent A: | 99% water/1% acetonitrile; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS000725 |
| Analysis ID: | AN000824 |
| Instrument Name: | ABI Sciex API 5000 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
