The 8th Mass Spectrometry CVG Annual Symposium - Full Day Event!!!
Presentation No. 1 - Accurate and Sensitive All-Ions Quantitation Using a New Ultra High Resolution LCMS and its Application to Endogenous Metabolite Profiling
Mark Sanders1; Kevin J. Mchale1; Chunang (christine) Gu2; Petia Shipkova3
1Thermo Fisher Scientific, Somerset , NJ; 2ThermoFisher Scientific, San Jose, CA; 3Bristol Myers Squibb, Princeton, NJ
Novel Aspect: Accurate and sensitive multi-component quantitation achieved by utililizing high resolution accurate mass LC/MS data
Introduction: Endogenous metabolite profiling (metabolomics) is an extreme example of multi-component quantitation. Even though in most cases the quantitative comparisons between samples are relative, the key to a successful study is high quality quantitative results. In some studies the differences between groups can be quite subtle and high analytical reproducibility is required to identify potential biomarkers . Once potential biomarkers have been found, more rigorous, absolute quantitative assessments are usually made to validate the initial observations. Here we demonstrate the use of ultra high resolution LCMS to provide sensitive, high quality quantitative data. This then provides a simple, relatively unbiased and highly quantitative assessment of metabolomics samples and negates the need for multiple MS platforms.
Methods: Protein precipitated plasma was analyzed by full-scan MS acquired from m/z 85-1000 at resolutions up to 100,000 FWHM on the Exactive (Thermo Scientific) orbitrap mass spectrometer. Separation was achieved on a 2.1x150 mm, 1.9 um Hypersil Gold column via a 15 min uHPLC gradient elution method. Data analysis was performed using Sieve software and statistical approaches to identify potential biomarkers. The identities of the components of interest were confirmed using fragmentation data generated by passing ions through a collision cell (HCD cell) on the Exactive instrument. The quantitative performance was validated using stable labeled standards spiked into rat plasma and analyzed with the same chromatography on a TSQ Quantum Access (Thermo Scientific) triple quadrupole mass spectrometer.
Results: High resolution LC/MS platforms are often used for metabolomic screening to allow for a relatively unbiased view of the sample and the ability to deconvolute the many components of a complex biological sample. However, the triple quadrupole mass spectrometer is considered the gold standard for quantitation and so typically, the validation of potential biomarkers are performed with LC/MS SRM techniques. The problem with this approach is that MS/MS methods need to be developed for each analyte of interest and that as the number of analytes increases the advantages of using a triple quadrupole diminish. The move to smaller SRM dwell times to accommodate more components across narrow uHPLC peaks compromises both sensitivity and precision. In addition, the more analytes the more labor intensive the SRM method development becomes. In contrast, the instrument set up for high resolution quantitation is very simple, with a full-scan over the mass range of interest and no prior knowledge of the analytes needed. Selectivity for the quantitative measurement is provided by high resolution MS and the use of narrow mass windows (3ppm) around the analyte of interest. This provides a high degree of selectivity without losing ion current, which occurs when MS/MS techniques are used. Quantitative performance was comparable to that of a triple quadrupole when multiple analytes were monitored. A pooled control sample was used to ensure sample integrity throughout the study and comparison of this with individual animal data gave a comparison between analytical and biological variability. For a series of amino acids the analytical variability (~5%) was well within the biological variability observed (~15%).
Using this approach the metabolomics experiment is greatly simplified and all stages of the workflow from screening, to identity confirmation, to absolute quantitative measurements can be performed on a single ultra high resolution LC/MS platform.
