Fast sampling method for mammalian cell metabolic analyses using liquid chromatography–mass spectrometry

Abstract

Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a -20 °C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LC-MS method is applied.

Metabolite Measurement: Pitfalls to Avoid and Practices to Follow

“…Two main problems can arise during quenching: ( a ) perturbation of metabolite levels in the harvesting steps or ( b ) incomplete or insufficiently fast termination of enzyme activity during quenching. For analysis of primary metabolites from cellular and tissue samples, rapid quenching is necessary as the turnover rate can be on the order of 1 s for compounds like ATP and glucose 6-phosphate (8–12). …”

“…Existing literature is ambiguous about the best solvent systems for metabolite extraction (12, 25–31). This reflects that many metabolites, including amino acids, Krebs cycle intermediates, and glycolytic intermediates, are adequately extracted using a variety of solvents.…”

Training in metabolomics research. I. Designing the experiment, collecting and extracting samples and generating metabolomics data

“…For adherent cells grown on culture plates, an effective procedure is to rapidly remove the culture medium with a Pasteur pipet attached to a vacuum line. The cells are immediately washed with ice‐cold, isotonic physiological saline which after swirling for a few seconds is also aspirated . The washed cells can either be instantly frozen with liquid N 2 to be extracted at a later time or immediately extracted with methanol pre‐cooled to dry ice temperature (−43 °C).…”

Training in metabolomics research. I. Designing the experiment, collecting and extracting samples and generating metabolomics data

“…The cells are immediately washed with ice-cold, isotonic physiological saline which after swirling for a few seconds is also aspirated. [36] The washed cells can either be instantly frozen with liquid N 2 to be extracted at a later time [37] or immediately extracted with methanol pre-cooled to dry ice temperature (−43 °C). These steps serve to 'freeze' metabolism.…”