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.

Metabolomics 20 years on: what have we learned and what hurdles remain?

“…Current plant metabolomics strategies are reliant on either mass spectrometry‐ or nuclear magnetic resonance (NMR)‐based approaches. A number of detailed protocols have been published (Lisec et al ., ; De Vos et al ., ; Kruger et al ., ; Tohge and Fernie, ), alongside several excellent technical reviews (Fiehn, ; Sumner et al ., ; Kopka et al ., ; Cajka and Fiehn, ; Fiehn et al ., ; Lu et al ., ). In response, we will restrict ourselves here to just a brief outline of the relative advantages and disadvantages of the three major methods currently in use, namely gas‐chromatography mass‐spectrometry (GC‐MS), liquid‐chromatography mass‐spectrometry (LC‐MS) and NMR.…”

Multiplex SERS Detection of Metabolic Alterations in Tumor Extracellular Media

“…[ 10 ] NMR allows quantifying most components in biological fluids with no need for elaborate sample preparation; however, the sensitivity of NMR spectroscopy remains a weak point, as compared, e.g., to LC‐MS. [ 11 ] Hence, the development of alternative label‐free methods to rapidly detect multiple tumor‐secreted metabolites in extracellular media is required toward understanding metabolic interactions in the tumor niche. [ 12 ]…”

Low catalytic activity is insufficient to induce disease pathology in triosephosphate isomerase deficiency

“…Immediately following dissection, tissues from 20‐week‐old male mice were freeze clamped in liquid nitrogen and stored at −80°C. Tissues were ground to a fine powder over liquid nitrogen and extracted in a mix of chloroform, methanol, and water in a ratio of 40:40:20 with 0.1 M formic acid, as previously described …”

“…Immediately following dissection, tissues from 20-week-old male mice were freeze clamped in liquid nitrogen and stored at −80 C. Tissues were ground to a fine powder over liquid nitrogen and extracted in a mix of chloroform, methanol, and water in a ratio of 40:40:20 with 0.1 M formic acid, as previously described. 40 The samples were analyzed by tandem mass spectrometry on an Agilent 1290 liquid chromatography system coupled to an Agilent 6470 triple quadrupole mass spectrometer. The LC-MS/MS method used was based on the Agilent Metabolomics dMRM Database and Method with a shortened chromatography.…”