Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer

Abstract

We present a liquid chromatography-mass spectrometry (LC-MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and unanticipated metabolites in parallel, with a focus on water-soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water-methanol gradient on a C18 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 to 1000 m/z at 1 Hz and 100,000 resolution in negative ion mode on a stand alone orbitrap ("Exactive"). The median limit of detection, across 80 metabolite standards, was 5 ng/mL with the linear range typically >or=100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker's yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose (13)C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL215C), we observed accumulation of an ion of m/z 128.0351, which we subsequently confirmed to be oxoproline, resulting in annotation of YKL215C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling.

Figure 1

Negative ion mass spectrum of arginine standard at 10 µg/mL showing the base peak at m/z 173.1044, and its natural isotope peaks with one ¹⁵N (m/z 174.1012) and one ¹³C (m/z 174.1077). The latter two were not resolved at a resolution setting of 10,000 or 25,000, partially separated at 50,000, and well separated at 100,000.

Figure 2

Overlay of extracted ion chromatograms of selective metabolites from a S. cerevisiae extract.

Figure 3

Relative labeling percentage of two representative metabolites as a function of time: fructose-1,6-bisphosphate (FBP, top panel), and citrate (bottom panel). For the purpose of simplicity, the following labeled forms were not plotted: ¹³C₁- and ¹³C₂-labeld FBP which has a maximum percentage of 1.7% and 1.4%, respectively, and ¹³C₁-labeled citrate which has a maximum percentage of 1.3%. The standard deviations for N=2 were plotted only for the unlabeled forms.

Figure 4

Chromatogram traces of m/z slice 128.0347–128.0360 (128.0353 ± 5 ppm) from extracts of wild type yeast and ykl215c (oxp1) deletion mutant yeast (each with four biological replicates). The replicates of the same strain give similar results, while there is a > 3-fold difference for the features at 7.2 min between the WT and mutant strains (p=0.0005 by T-test). The feature was identified as 5-oxoproline (see text for details).