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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Combined analysis:
Analysis ID | AN000824 |
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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 |