Metabolic Profiling of Klebsiella oxytoca: Evaluation of Methods for Extraction of Intracellular Metabolites Using UPLC/Q-TOF-MS

Abstract

The global pool of intracellular metabolites is a reflection of all the metabolic functions of an organism. In the absence of in situ methods capable of directly measuring metabolite pools, intracellular metabolite measurements need to be performed after an extraction procedure. In this study, we evaluated the optimization of technologies for generation of a global metabolomics profile for intracellular metabolites in Klebsiella oxytoca. Intracellular metabolites of K. oxytoca were extracted at the early stationary phase using six different common extraction procedures, including cold methanol, boiling ethanol, methanol/chloroform combinations, hot water, potassium hydroxide, and perchloric acid. The metabolites were subsequently collected for further analysis, and intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. During analysis, the stability of metabolites extracted using cold methanol was clearly higher than that obtained by other extraction methods. For the majority of metabolites, extracts generated in this manner exhibited the greatest recovery, with high reproducibility. Therefore, the use of cold ethanol was the best extraction method for attaining a metabolic profile. However, in another parallel extraction method, perchloric acid may also be required to maximize the range of metabolites recovered, particularly to extract glucose 1-phosphate and NADPH.

Salivary biomarkers in cancer detection

“…It allows measuring the levels of endogenous metabolites, thus enabling biomarker discovery [28]. The endogenous metabolites, including nucleic acids, lipids, amino acids, peptides, vitamins, organic acids, thiols, and carbohydrates, represent a valuable tool for detection of biomarkers for various diseases and monitoring disease progression [29, 30]. …”

Metabolomic analysis of the food-borne pathogen Campylobacter jejuni: application of direct injection mass spectrometry for mutant characterisation

“…Similarly, washing steps were avoided to prevent metabolite loss but contamination of cell pellets with residual medium will be the same for each sample compared and medium-only controls were also run. Although the efficiency of extraction techniques is organism specific (Faijes et al 2007; Meyer et al 2013), comparative studies have shown cold methanol based methodologies to be among the most efficient for extracting a wide range of metabolites, in a broad spectrum of bacteria (Maharjan and Ferenci 2003; Park et al 2012). For this reason a cold methanol:chloroform based extraction protocol was adopted here (adapted from Overy et al (2005)).…”

Metabolomic Analysis of Campylobacter jejuni by Direct-Injection Electrospray Ionization Mass Spectrometry

“…subjected to the same quenching and extraction conditions, valid comparisons are possible. The efficacy of metabolite extraction techniques have been shown to be organism specific (8), but many comparative studies have shown cold methanol based methodologies to be the most efficient for extracting the broadest range of metabolites (8,9,10). For this reason a simple cold methanol based extraction protocol was adopted in our work, enabling high reproducibility and extraction of a broad range of metabolites (1,11).…”