The “Exactive” is a stand-alone orbitrap mass spectrometer that is designed for high resolution full scans. Ions formed by electrospray ionization are transferred to a curved linear trap (C-Trap), from which they are injected into the orbitrap for mass analysis. Mass resolution depends on scan speed. At m/z 200, resolution is 10,000 at 10 Hz; 25,000 at 4 Hz; 50,000 at 2 Hz; and 100,000 at 1 Hz. The effect of the Exactive’s resolving power on analytical results has been demonstrated recently 20
. We find that the higher resolution settings offer important benefits, as they frequently help to separate overlapping ion peaks. As an example, shows the mass spectrum of arginine (purified standard), with a focus on the natural isotope peaks containing one 15
N nucleus (m/z 174.1012) versus one 13
C nucleus (m/z 174.1077). These two peaks merge at a resolution setting of 10,000 and 25,000, partially resolve at 50,000, and separate fully at 100,000. The Exactive’s ability to separate 15
N- and 13
C-isotopomer peaks is useful for isotope tracer experiments.
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 (more ...)
Like any trap instrument, the performance of Exactive is affected by the space-charge effect 37
. If there are too many ions inside the trap, these will lead to distortion of the electric field and compromise the instrument performance. The Exactive has an Automatic gain Control (AGC) function which controls the duration of ion injection into the orbitrap to maintain an optimum total number of ions (the AGC target value). When using AGC, the instrument alternates between pre-scans and analytical scans. When the pre-scan finds a large total ion current (TIC), the injection time for the subsequent analytical scan is reduced. The user selects between three AGC target values: 3e6 for a high dynamic range scan, 1e6 for a balanced scan, and 5e5 for the best mass accuracy. Here we used the 3e6 target value to maximize quantitative performance, with the 5e5 target value useful for improving mass accuracy when aiming to identify unanticipated metabolites. To mitigate further the space-charge effect caused by anions in the cell culture media (phosphate and sulfate), we modulated the instrument’s molecular weight scan range to reduce accumulation of these ions in the orbitrap during the chromatographic internals where they elute (see Materials and Methods for details).
LC-MS method development and validation
Luo et al. originally described an LC-MS/MS method for metabolomics that coupled reversed phase HPLC with tributylamine as an ion-pairing reagent via electrospray ionization to triple quadrupole MS/MS 23
. Run time was 80 min. We subsequently modified the gradient to reduce the running time to 50 min 24
. The column particle size was 4 µm and the flow rate 200 µL/min. Although briefer than the original method of Luo et al., this method was still undesirably slow. Efforts to expedite analysis, however, ran up against the need to for relatively slow compound elution to accommodate a large number of selected reaction monitoring scan events while retaining adequate coverage of individual chromatogram peaks. This is a typical problem when using triple-quadrupole instruments for “omic” analysis.
In the current method, we aimed to take advantage of the full scan capability of the Exactive orbitrap to expedite analysis. To this end, we selected a column particle size of 2.5 µm, which, without changing flow rate, enabled us to reduce the total running time to 25 min. Most of the resulting chromatogram peaks have a width of ~ 15 s, resulting in 15 data points across the peaks when scanning at 1 Hz (100,000 resolution). The maximum pump pressure during the LC run is ~ 300 bar, which can be obtained on high quality HPLC systems, as well as UPLC systems.
In Supporting Information, Table S-1
we summarize the results of LC-MS method validation for 87 metabolite standards. The median limit-of-detection (LOD) is 5 ng/mL with 63 compounds (72%) having a LOD ≤ 10 ng/mL. The compounds with a higher LOD are mostly nucleotide di- and triphosphates and coenzyme A derivatives. All the compounds gave a linear response (R2
> 0.98) over a concentration range of at least 10-fold, with 68 compounds (78%) showing an R2
> 0.98 over at least a 100-fold concentration range. The quantitative reproducibility (intra-day) was determined at a compound concentration of 100 ng/ml. For the 82 metabolites with an LOD ≤ 100 ng/mL, median RSD is 8.2% with 74 compounds (90%) showing a RSD < 20%.
We also evaluated mass accuracy using external calibration. For the 87 metabolites studied, 86 (99%) showed a ppm error of < 5 ppm with a median of −0.29. Mass accuracy is particularly good at the low mass region where most of compounds of interest appear. Mass accuracy can likely be further improved by using internal calibration.
Finally, we tested the method on an extract of Baker’s yeast, Saccharomyces cerevisiae. We detected 137 known metabolites, with the observed signals (peak heights), as well as RSDs (intra-day) listed in . The median RSD is 7.6%, indicating good reproducibility in real samples. Metabolites detected include amino acids, carboxylic acids, sugar phosphates, nucleotides, and coenzyme A derivatives, indicating the ability of the method to quantitate much of the core metabolome. Ion-specific chromatograms corresponding to selected metabolites are shown in .
Retention time, mass accuracy, signal intensity, and quantitative reproducibility for 137 metabolites detected from Saccharomyces cerevisiae extract
Overlay of extracted ion chromatograms of selective metabolites from a S. cerevisiae extract.
Kinetic flux profiling
Upon switching from unlabeled to isotope-labeled nutrient, intracellular metabolites becomes labeled, with the rates of labeling depending on the proximity of the metabolite to the labeled nutrient in the metabolic network and the flux through the metabolite. Accordingly, labeling dynamics (kinetic flux profiling) can be used to probe metabolic network structures, as well as to quantitate metabolic fluxes 27, 29
In our previous studies, this was done on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode. When examining many metabolites, the MRM-based approach is cumbersome, especially for metabolites that can be partially labeled in many different ways, as each partially labeled form requires its own MRM scan. Moreover, selecting the appropriate product ion to monitor the partially labeled forms is not always straightforward 27, 38
. Using high resolution full scan MS, labeled metabolites can be identified based on their accurate masses. With this approach, we are able to monitor the labeling patterns of a large number of metabolites in parallel. Here we show results obtained upon switching E. coli
from unlabeled to uniformly 13
C-labeled glucose for two representative compounds: fructose-1,6-bisphosphate (FBP) in glycolysis and citrate in TCA cycle (). Comparable quality of labeling data is obtained for most compounds. The full data, in tandem with relevant network-level analyses, will be described elsewhere.
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 (more ...)
Consistent with its position just downstream of glucose in the high-flux pathway of glycolysis, FBP labels quickly, with a half-time of ~ 2 min. Initially, FBP accumulates in two labeled forms: fully labeled and 3 × 13
C-labeled. The fully labeled form is consistent with passage of glucose down glycolysis, whereas the three-labeled form is consistent with the reverse aldolase reaction joining together a fully labeled with an unlabeled triose phosphate (dihydroxyacetone phosphate or glycealdehyde-3-phosphate). The initial accumulation of three-labeled FBP at ≥ 50% of the rate of fully labeled FBP implies the reverse aldolase flux is a substantial fraction of the net glycolytic flux, i.e., that the aldolase reaction is near to equilibrium in glucose-fed E. coli
. This observation is consistent with recent thermodynamic analysis of the glycolytic pathway 39
Citrate is a six-carbon metabolite formed by the condensation of the acetyl carbons of acetyl-CoA with the four-carbon TCA cycle compound oxaloacetate. Oxaloacetate can be generated in E. coli either by carboxylation of the three-carbon glycolytic compound phosphoenolpyruvate (“anapleurosis”) or by turning of the TCA cycle. These sets of reactions lead to formation of citrate with 2, 3, 4, 5, or 6 labeled carbon atoms. Initially, 2 × 13C-labeling (from acetyl-CoA) and 3 × 13C-labeling (from anapleurosis) dominate, with subsequent rises in 4, 5, and 6 carbon labeling. After 30 min, the most abundant form is 5 × 13C-labeled, consistent with a 3 × 13C-labeled oxaloacetate (formed by the condensation of unlabeled carbon dioxide from the environment with labeled phosphoenolpyruvate) being joined with two labeled carbon atoms from acetyl-CoA. These data indicate that the anapleurotic flux in E. coli is larger than the flux associated with complete turning of the TCA cycle: complete turning of the TCA cycle would lead to 4 × 13C-labeling (from 2 × acetyl-CoA) exceeding 3 × 13C-labeling and 5 × 13C-labeling (from anapleurosis) at early time points; similarly, complete turning of the TCA cycle would lead to complete citrate labeling (rather than five carbon labeling) at late time points.
Discovery metabolite profiling
Even for well-studied organisms like E. coli
and yeast, the functions of many genes remain unknown. For metabolic enzymes, comparison of the metabolome of wild-type and gene deletion strains provides a powerful tool for gene function elucidation. Enzyme knockout typically leads to the elevation of the enzyme’s substrates and/or the depletion of its products. This approach has been previously used to assign enzyme functions in mammals 40
. Its success relies on having an analytical method for metabolome profiling which is adequate to quantitate the enzyme’s substrates and/or products. The method should preferably be untargeted, to enable discovery also of novel metabolites, enzymatic activities, and pathways.
Here we used the present LC-MS method to investigate the function of the gene YKL215C, whose role in yeast was previously unknown. Untargeted metabolite profiling of extracts of the wild type and knockout strains revealed >10,000 mass spectral features. Three m/z features with ≥ 3-fold differences between all mutant and control strains were identified, each of which appeared at the same retention time, 7.2 min, with the strongest signal associated with m/z 128.0351 (). The other two features are at m/z 279.0593 and m/z 355.0454, with a relative signal intensity of 10% and 0.7% of the feature at m/z 128.0353. When searched against the KEGG database, the compound with m/z 128.0351 in negative ion mode matched the exact mass of 5-oxoproline (neutral formula C5H6NO3, m/z 128.0353 in negative mode) with < 2 ppm mass accuracy.
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, (more ...)
Additional experiments were performed to confirm the compound’s identity. First, when fed [U-13C]-glucose or 15N-ammonia, we observed the disappearance of ion of m/z 128.0351. Instead, at the same retention time, we observed the accumulation of ions with m/z 133.0516, and m/z 129.0317, respectively. The masses match those of 5 × 13C oxoproline (expected m/z 133.0521, 3.8 ppm error), and 1 × 15N oxoproline (expected m/z 129.0323, 4.6 ppm error), consistent with 5-oxoproline containing five carbons and one nitrogen. Secondly, we spiked the cellular extracts with authentic standard of 5-oxoproline at concentrations of 0.5 and 2.5 µg/mL. The peak signal corresponding to m/z 128.0351 with a retention time of 7.2 minute increased with the concentration of standard added.
The other compounds that were elevated in the YKL215C deletion strain had exact masses of m/z 279.0593, and m/z 355.0454, and both co-eluted with 5-oxoproline, but with smaller ion intensity. Both also increase upon the addition of the 5-oxoproline standard to a metabolite extract, consistent with their being adducts of 5-oxoproline, although we remain unsure of their precise identities.
Based on these data, we hypothesized that Ykl215c is an oxoprolinase. Consistent with this, subsequent investigation revealed that YKL215C shares 48% sequence identity to the verified M. musculus ATP-hydrolyzing 5-oxoprolinase (gene name Oplah). Based on these results, and following the nomenclature of the oxoprolinase genes in plants, we have registered the gene name in the Saccharomyces Genome Database (SGD) as OXP1. Thus, the present method has been successfully employed, without the need for MS/MS, for untargeted metabolite profiling, resulting in improved annotation of the genome of Baker’s yeast.
Comparison with triple-quadrupole instrument
The market price of the “Exactive” benchtop orbitrap instrument is comparable to that of modern triple-quadrupole instruments. Accordingly, a practical question regards their relative advantages and disadvantages for metabolomic analysis (). Both techniques are suitable for targeted analysis. Triple quadrupole instruments use MS/MS (multiple reaction monitoring, MRM) to achieve a high degree of analyte specificity, even in complex biological samples. On the downside, pre-optimization is required to determine appropriate MRM parameters. The measured compounds are limited to those targeted by MRM events programmed in the method. In addition, quantitative performance decreases with increasing number of MRM scan events, since each scan event takes a fixed time (so-called “scan time” or “dwell time”).
A comparison of triple quadrupole MS/MS and “Exactive” orbitrap full scan MS for metabolomics.
The “Exactive” orbitrap mass analyzer, on the other hand, detects ions solely using high resolution accurate mass. The high resolution is critical to obtaining adequate analyte specificity in complex biological samples without MS/MS. Pre-optimization is not required. Rather a generic full scan method can be used, looking for everything in the appropriate scan range. The number of compounds that can be detected is virtually unlimited. This is particularly useful when following many partially labeled metabolites in isotope tracer experiments. Quantitative performance is generally not affected by the number of metabolites to be detected, as long as the total number of ions entering orbitrap at any given moment does not induce space charge effects 37
. A major advantage of the orbitrap is its usefulness also for untargeted analysis. While accurate mass alone is generally not sufficient to identify an unknown compound, it is a critical first step towards such a goal.
In our hands, the quantitative performance of both instrument types is quite similar. Both show sensitivity in the ng/mL range, linear response over 2–3 orders of magnitude, and reasonable reproducibility. Our finding that both instrument types offer similar quantitative performance for metabolomics is consistent with recent literature reaching a similar conclusion in the areas of pharmacokinetic analysis 19
and small molecule quantitation 17
In terms of specificity, each instrument type has its strengths and weaknesses. The orbitrap’s high resolving power offers certain advantages compared to triple quadruple MS/MS. For example, lysine (m/z 145.0983) and glutamine (m/z 145.0619), which have very similar MS/MS spectra, can be distinguished without the need for LC separation based on their exact masses. Similarly, IMP (m/z 347.0398) can also be distinguished from 13C1-labeled AMP (m/z 347.0592); as AMP is typically more abundant than IMP in cell extracts, this proves a practical virtue in metabolomic analysis even in the absence of isotope labeling. On the other hand, full scan alone can never distinguish isomers; a second dimension separation such as LC is needed. In contrast, it may be possible to distinguish isomers by MRM alone. For example, citrate can be detected using selected reaction monitoring (SRM) transition m/z 191 (C6H7O7−) ➔ 87 (C3O3H3−) at 18 eV, and isocitrate can be detected using SRM m/z 191 (C6H7O7−) ➔ 117 (C4O4H5−)at 18 eV. Also, certain interferences can be better separated by MS/MS than exact mass. For example, in the present method, an unknown interference at m/z 89.0244 typically masks the signal for lactate (m/z 89.0244). Although uncommon, such cases highlight the utility of having multiple different MS techniques available.