Development of an LC-MS Targeted Metabolomics Methodology to Study Proline Metabolism in Mammalian Cell Cultures

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.

Prolidase Stimulates Proliferation and Migration through Activation of the PI3K/Akt/mTOR Signaling Pathway in Human Keratinocytes

“…4.12. LC-MS-Based Quantitative Analysis LC-MS analysis of proline concentration in HaCaT cells was conducted according to the method of Klupczynska et al [52]. Briefly, cells were harvested by scraping in ice-cold methanol with stable isotopically labeled proline (25 µM, d3-proline; Sigma Aldrich, Saint Louis, MO, USA) as an internal standard.…”

Platelet-Rich Plasma Promotes the Proliferation of Human Keratinocytes via a Progression of the Cell Cycle. A Role of Prolidase

“…Proline concentration in culture HaCaT cells was measured with the use of the method published by Klupczynska et al [ 46 ]. Samples were analyzed using Agilent 1260 Infinity HPLC system coupled to Agilent 6530 Q-TOF mass spectrometry detector (Agilent Technologies, Santa Clara, CA, USA) with electrospray ionization as an ion source in positive ionization mode.…”

PRODH/POX-Dependent Celecoxib-Induced Apoptosis in MCF-7 Breast Cancer

“…LC–MS analysis of proline was conducted according to the method of Klupczynska et al [ 34 ]. Methanol-extracted proline was subjected to Agilent 1260 Infinity HPLC system coupled to Agilent 6530 Q-TOF mass spectrometry detector (Agilent Technologies, Santa Clara, CA, USA) with electrospray ionization as an ion source in positive ionization mode.…”