HRAM Mass Spectrometry for Metabolomics

High resolving power is especially useful during isotope labeling metabolomics experiments. One particular example involves the use of labeled amines such as [15N]glutamine in chronic pain test subjects. Here, the labeled glutamine and endogenous glutamate both elute on the same reversed-phase LC column and cannot be resolved by using a resolution of 20,000 because the monoisotope of labeled glutamine (A0) is overlapped by the second isotope of glutamate (A1). As a result, quantitative measurement becomes a challenge.

Using high resolution MS, such as that found with Orbitrap mass analyzer technology, the instrument can be set at a resolution of 100,000. This leads to the resolution of two amine peaks instead of one, and each peak can subsequently be characterized.

For quantitative experiments, unambiguous results can only be obtained if sufficient resolving power is used to separate the target metabolite from any possible interferences. If the resolving power of the instrument is insufficient, false positive or false negative signal responses will be generated.  

Metabolomics is and always has been a quantitative science. This is because there are order-of-magnitude differences in the concentrations of endogenous metabolites. As such, a mass spectrometer must be able to detect these concentration variances in order to generate data that reflect a comprehensive and meaningful view of the biological condition under study. With Orbitrap mass analyzer technology, analyte quantitation can be performed down to extremely low femtomole levels. Furthermore, analytes ranging over five orders of linear dynamic range can be easily detected, with the reslting data still achieving small coefficient of variation (CV) values.

Because of its high sensitivity and selectivity, mass spectrometry techniques have become the method of choice for metabolomics research. To uncover meaningful answers from a large number of data sets, which is the norm with metabolomics studies, the mass spectrometer must provide accurate and reproducible data from run to run, and without the need for internal calibration.

Despite the need for fast data acquisition during metabolomics experiments, there must still be no compromise in the top quality HRAM spectra used in MS or MSⁿ modes for confident compound identification.

Due to metabolomics studies featuring complex samples as well as sample contaminants, having a narrow precursor isolation window is vital to ensuring that the MS/MS spectra do not also contain peaks from background interferences. Such interferences, if present, then make target compound identification more difficult.

One solution to this challenge is to maintain a tight isolation window. In the example below, a tight 1-4 amu isolation window keeps transmission losses at a minimum while still limiting chemical noise interference. A high quality MS/MS spectrum of bovine heart total lipid extract (1 mg on column) is generated, with a 1 amu isolation width for enhanced specificity.

With metabolomics compounds having diverse physico-chemical properties, a combination of separation techniques (GC, IC and LC reversed phase/HILIC) can help achieve a more comprehensive view of different biological states. Additionally, greater spectral coverage can be achieved by using both positive and negative ionization modes on the mass spectrometer. This is feasible only if fast polarity switching is also used, because faster polarity switching enables a more productive separations time scale during a single run.

Spectral libraries of various MSⁿ levels offer spectral identification certainty. Different fragmentation modes and collision energies must be available for this approach to work.