X13CMS: global tracking of isotopic labels in untargeted metabolomics

Abstract

Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X(13)CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X(13)CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X(13)CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X(13)CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X(13)CMS with an analysis of cultured rat astrocytes treated with uniformly labeled (U-)(13)C-glucose during lipopolysaccharide (LPS) challenge. Our results show that out of 223 isotopologue groups enriched from U-(13)C-glucose, 95 have statistically significant differential labeling patterns in astrocytes challenged with LPS compared to unchallenged control cells. Only two of these groups overlap with the 32 differentially regulated peaks identified by XCMS, indicating that X(13)CMS uncovers different and complementary information from untargeted metabolomic studies. Like XCMS, X(13)CMS is implemented in R. It is available from our laboratory website at http://pattilab.wustl.edu/x13cms.php .

Figure 1

X¹³CMS workflow. Samples representing control and perturbed conditions are divided into two groups, with one receiving an isotopically labeled substrate at a defined fraction of the total pool of that substrate and the other receiving the same substrate in a completely unlabeled or natural isotope abundance form. Metabolite extracts and LC/MS profiling data are collected on all samples and processed through XCMS to detect features and perform retention-time alignment. The features tables representing unlabeled and labeled samples are then paired off by biological condition and submitted to the getIsoLabelReport() routine of X¹³CMS to detect features that have been enriched for the isotope label. To compare labeling patterns between conditions, the isotope labeling reports are processed with getIsoDiffReport().

Figure 2

Validating isotopologue retrieval by X¹³CMS. MS/MS spectra at a collision energy of 10 V for (a) glutamic acid standard, (b) base isotopologue of the 146 m/z group in unlabeled astrocytes, (c) base isotopologue of the 146 m/z group in U-¹³C-glucose-labeled astrocytes (top) and 148 isotopologue (bottom) in labeled astrocytes. Star indicates precursor ion. Intensities are expressed relative to the most intense fragment. Detection of (d) U-¹³C-lactate and (e) alpha-¹⁵N-glutamine spiked into unlabeled, LPS-stimulated astrocytes.

Figure 3

Differential labeling analysis of control and LPS-challenged rat astrocytes. (a) Experimental design. (b) All label-enriched isotopologue groups identified in control and LPS-stimulated cells. (c) Isotopologue groups identified as being differentially labeled in control versus LPS-stimulated cells. (d) Extracted ion chromatograms for the isotopologue group with base m/z of 133.013. (e) Enrichment of each isotopologue of the 133.013 m/z group in labeled cells compared to unlabeled cells of each culture condition (control vs LPS-stimulated).

Figure 4

Comparison of outputs from untargeted isotope labeling analysis and label-free untargeted metabolomics.