Abstract
A growing interest in metabolomics studies of cultured cells requires development not only untargeted methods capable of fingerprinting the complete metabolite profile but also targeted methods enabling the precise and accurate determination of a selected group of metabolites. Proline metabolism affects many crucial processes at the cellular level, including collagen biosynthesis, redox balance, energetic processes as well as intracellular signaling. The study aimed to develop a robust and easy-to-use targeted metabolomics method for the determination of the intracellular level of proline and the other two amino acids closely related to proline metabolism: glutamic acid and arginine. The method employs hydrophilic interaction liquid chromatography followed by high-resolution, accurate-mass mass spectrometry for reliable detection and quantification of the target metabolites in cell lysates. The sample preparation consisted of quenching by the addition of ice-cold methanol and subsequent cell scraping into a quenching solution. The method validation showed acceptable linearity (r > 0.995), precision (%RSD < 15%), and accuracy (88.5–108.5%). Pilot research using HaCaT spontaneously immortalized human keratinocytes in a model for wound healing was performed, indicating the usefulness of the method in studies of disturbances in proline metabolism. The developed method addresses the need to determine the intracellular concentration of three key amino acids and can be used routinely in targeted mammalian cell culture metabolomics research.
Highlights
Culture cell metabolomics is a field of metabolomics that has the potential to reflect the phenotype of any cell and enhance our knowledge of cellular biochemistry, functions, and response mechanisms [1].Cell metabolomics investigations aim to characterize profiles of small molecular weight compounds and usually focus on capturing changes associated with different signals or perturbations, e.g., gene mutations, pharmacological interventions, exposure to a toxin or stress agents
The method was validated and successfully applied to a pilot study of alterations of proline metabolism in spontaneously immortalized human keratinocytes HaCaT cell line. The results from both a series of validation tests and a pilot study on cell lines confirmed that the developed methodology used routinely in targeted mammalian cell culture metabolomics research
It constitutes a reliable tool in targeted studies of proline metabolism in cell cultures and can be expanded by adding new metabolites related to the proline metabolic axis, belonging to amino acids
Summary
Culture cell metabolomics is a field of metabolomics that has the potential to reflect the phenotype of any cell and enhance our knowledge of cellular biochemistry, functions, and response mechanisms [1]. There is a need for the application of additional sample processing steps, such as cell metabolism quenching and metabolite extraction, compared to the use of biofluids that usually require only protein precipitation and dilution. The study aimed to develop a robust and easy-to-use targeted metabolomics method for the determination of the intracellular level of proline and the other two amino acids closely related to proline metabolism: glutamic acid and arginine. The method was validated and successfully applied to a pilot study of alterations of proline metabolism in spontaneously immortalized human keratinocytes HaCaT cell line The results from both a series of validation tests and a pilot study on cell lines confirmed that the developed methodology used routinely in targeted mammalian cell culture metabolomics research.
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