Metabolite Measurement: Pitfalls to Avoid and Practices to Follow

Abstract

Metabolites are the small biological molecules involved in energy conversion and biosynthesis. Studying metabolism is inherently challenging due to metabolites' reactivity, structural diversity, and broad concentration range. Herein, we review the common pitfalls encountered in metabolomics and provide concrete guidelines for obtaining accurate metabolite measurements, focusing on water-soluble primary metabolites. We show how seemingly straightforward sample preparation methods can introduce systematic errors (e.g., owing to interconversion among metabolites) and how proper selection of quenching solvent (e.g., acidic acetonitrile:methanol:water) can mitigate such problems. We discuss the specific strengths, pitfalls, and best practices for each common analytical platform: liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and enzyme assays. Together this information provides a pragmatic knowledge base for carrying out biologically informative metabolite measurements.

Keywords: accuracy; mass spectroscopy; metabolite extraction; metabolomics; metabonomics; stability.

Figure 1

Metabolite interconversion owing to incomplete quenching of enzymatic activity is prevented by 0.1 M formic acid (FA). (a) HEK293 T cells were grown in ¹³C₆-glucose media to completely label glycolytic intermediates. Unlabeled 3-phosphoglycerate (3PG) was added to the extraction solvent of 80:20 methanol:H₂O (−70°C), which contained no FA, or 0.02 M FA, or 0.1 M FA. Phosphoenolpyruvate (PEP) can be made from 3PG through enolase activity. Extraction with 0.1 M FA eliminates unlabeled PEP made from the added 3PG standard. The same reaction occurs with endogenous (in this case, labeled) 3PG. Therefore, quenching without FA would greatly overestimate cellular PEP. (b) Yeasts were grown in ¹³C₆-glucose media to completely label cellular metabolites. Unlabeled ATP was added to the extraction solvent of 80:20 methanol:H₂O (−70°C), which contained no FA, or 0.02 M FA, or 0.1 M FA. Extraction with 0.1 M FA eliminated unlabeled ADP made from the added ATP standard. Therefore, quenching without FA would greatly overestimate ADP and underestimate the energy charge. Note that the FA should be neutralized after quenching to prevent acid-catalyzed degradation in the extract.

Figure 2

Comparison of two different extraction methods for polar metabolites from mouse liver samples. Mouse liver was ground using a cryomill, and 25 mg of ground tissue was then extracted in a 2 mL Eppendorf tube using the following methods: The first protocol (80% methanol) is to add 870 µL 80:20 methanol:water (v/v) at −70° C, vortex for 10 s, and place on dry ice for 20 min. The second protocol (addition of acetonitrile and formic acid) is to add 800 µL 40:40:20 acetonitrile:methanol:water + 0.1 M formic acid (solvent A) at −20°C, vortex for 10 s, place on ice for 2 min, add 70 µL 15% (w/v) NH₄HCO₃ in H₂O (solvent B), place in −20°C freezer for 20 min. For both protocols, samples were spun for 10 min at 16,000 × gravity, and the supernatant was analyzed by liquid chromatography-mass spectrometry (LC-MS). Control experiments (not shown) demonstrated that the difference in yields was due primarily to the solvent and not the temperature.

Figure 3

Effects of solvent evaporation on selected metabolites. Approximately 30 mg of mouse liver tissue was extracted using 1.2 mL 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid, followed by neutralization by NH₄HCO₃. The extract was divided into multiple 200 µL portions, dried using indicated methods, and redissolved in 200 µL of 10 mM NH₄HCO₃ in H₂O. Samples were analyzed by liquid chromatography-mass spectrometry (LC-MS), and the peak area of each compound was compared to those in the original extract. It was found that the majority of metabolites, including ATP, were not affected by the solvent evaporation process. By contrast, there was a significant decrease in NADPH and reduced glutathione (GSH) and an increase in NADP⁺ and oxidized glutathione disulfide (GSSG).

Figure 4

Measurement of the 6-phosphogluconate (M–H)⁻ ion by liquid chromatography-mass spectrometry (LC-MS) showing the effect of interfering compounds with similar mass from a mammalian cell extract. (a) Within a mass range of 275.01736 ± 20 ppm, two features were detected, one at ~12.8 min and one at ~13.3 min. (b) With a narrower range of 275.01736 ± 5 ppm, only the feature at ~13.3 min is observed. The feature at ~13.3 min is 6-phosphogluconate, with a measured mass of 275.0176 (+1.0 ppm error) and a retention time match to the authenticated standard. The other feature has a measured mass of 275.02167 (+16 ppm difference from 275.0174) and comes from an unknown interfering compound. Main panels a and b show mass-specific chromatograms. Insets show mass spectra with X-axis in units of m/z and the dashed line indicates the 6-phosphogluconate mass of 275.01736.

Figure 5

In-source fragments from electrospray ionization of citrate mimic other metabolites. (a) Mass spectrum taken at the retention time of citrate shows isotopic forms, adducts, and fragments: ¹³C₁-natural abundance peak (D), Na⁺ adduct (E), H₂O loss (C), CO₂ loss (B), and the loss of CO₂ + H₂O (A). (b) Mass-specific chromatograms show that citrate fragment ions mimic other metabolites. Channels refer to signals at the m/z of the stated metabolite molecular anion ±5 ppm. All of the peaks at 13.8 min are from citrate. The apparent itaconic acid peak at 13.1 min is from 2-hydroxyglutarate fragmentation.

Figure 6

Assessment of linearity. Plot of the liquid chromatography-mass spectrometry (LC-MS) peak areas (dark red dots) versus the concentration of the carnitine standard. The LC-MS method is reversed-phase chromatography on a C18 column coupled to a Q-Exactive plus mass spectrometer operating in positive ion mode. The signal is linear from 0.0005 to 1.2 µM and sublinear at higher concentrations.

Figure 7

Signal specificity and false discovery in one-dimensional (1D) ¹H-NMR-based metabolomics. Database searches using a hand-curated standard set (N = 270 common metabolites) from the Madison Metabolomics Consortium Database (MMCD; http://mmcd.nmrfam.wisc.edu) returns an average of 12 matches per 0.1 ppm region in a 1D ¹H NMR spectrum. This increases to an average of 137 metabolites per 0.1 ppm using the online tools available from the Human Metabolome Database (HMDB; http://www.hmdb.ca/) and 1,710 per 0.1 ppm for MMCD’s predicted chemical shift dataset (N = 19,070).

Figure 8

Evaluation of enzymatic assay for quantitation of NADPH/NADP⁺. (a) Consistent with the principle of the enzymatic assay, basic pH selectively degrades NADP⁺. 10 µM NADPH and 10 µM NADP in aqueous buffer containing 20mM nicotinamide, 20mM NaHCO₃, and 100mM Na₂CO₃ were heated at 60°C for the indicated times, and their concentrations were compared to the unheated samples by liquid chromatography-mass spectrometry (LC-MS). Note that modest degradation of NADPH is sufficient to alter NADP⁺ measurements, which are determined by subtraction. (b) Aqueous extraction without detergent using typical enzymatic assay conditions results in a dramatic loss of NADPH and a gain in NADP⁺, undermining utility of the enzyme assay. NADPH and NADP⁺ were measured from cultured HEK293T cells and mouse liver using two extraction methods: the suggested conditions of the enzymatic assay kit (20mM nicotinamide, 20mM NaHCO₃, 100 mM Na₂CO₃ in water) or our suggested extraction for LC-MS analysis (40:40:20 acetontrile:methanol:water + 0.1 M formic acid, followed by neutralization with NH₄HCO₃). Results were normalized to the LC-MS extraction buffer.