Influence of Drying Method on NMR-Based Metabolic Profiling of Human Cell Lines

Abstract

Metabolic profiling of cell line and tissue extracts involves sample processing that includes a drying step prior to re-dissolving the cell or tissue extracts in a buffer for analysis by GC/LC-MS or NMR. Two of the most commonly used drying techniques are centrifugal evaporation under vacuum (SpeedVac) and lyophilization. Here, NMR spectroscopy was used to determine how the metabolic profiles of hydrophilic extracts of three human pancreatic cancer cell lines, MiaPaCa-2, Panc-1 and AsPC-1, were influenced by the choice of drying technique. In each of the three cell lines, 40–50 metabolites were identified as having statistically significant differences in abundance in redissolved extract samples depending on the drying technique used during sample preparation. In addition to these differences, some metabolites were only present in the lyophilized samples, for example, n-methyl-α-aminoisobutyric acid, n-methylnicotimamide, sarcosine and 3-hydroxyisovaleric acid, whereas some metabolites were only present in SpeedVac dried samples, for example, trimethylamine. This research demonstrates that the choice of drying technique used during the preparation of samples of human cell lines or tissue extracts can significantly influence the observed metabolome, making it important to carefully consider the selection of a drying method prior to preparation of such samples for metabolic profiling.

Current progress in metabolomics of gestational diabetes mellitus

“…Due to the comprehensive nature of metabolomics, careful consideration of the following aspects is required: Selection and storage of the sample specimens, technology for the extraction of the metabolites, quality control of the analyses, and statistical methods to determine the metabolites undergoing significant change[ 30 ]. The basic procedures of metabolomics research consist of the experimental design, sample collection and pretreatment, metabolite extraction and detection, original data acquisition and processing, bioinformatics and statistical analysis, and annotation or identification of metabolites[ 31 ]. The main analysis workflow of the metabolomics analysis is shown in Figure 1 .…”

Dissolved organic metabolite extraction from high‐salt media

“…The process of sample drying can exert a powerful influence over the metabolite profiles determined by NMR 25 . SpeedVac drying involves the concentration of a sample by using a centrifugal vacuum evaporator 25 .…”

“…25 SpeedVac drying involves the concentration of a sample by using a centrifugal vacuum evaporator. 25 This is a high throughput method of sample drying that can be performed with temperature control in some instruments. Salt is concentrated to the point of saturation, so the sample is unsuitable for later analysis in some NMR probes.…”

“…25 In this process, the solvent sublimates from the sample, going directly from the solid to the gas phase. 25 Lyophilization has been shown to provide consistent results with strong reproducibility and minimal degradation of metabolites. 29 We transitioned to lyophilization in the middle of the DMSO-d 6 stage (in 3.7) of method development.…”

Methodological Developments for Metabolic NMR Spectroscopy from Cultured Cells to Tissue Extracts: Achievements, Progress and Pitfalls

“…An often-neglected detail in tissue extract preparation for NMR spectroscopy has been investigated in a rather recent paper by Petrova et al: effects on metabolite quantitation of the type of solvent removal chosen before re-dissolving the sample for NMR measurement [49]. The authors studied two techniques: speed vacuum centrifugal concentration (SpeedVac), and freeze-drying by sublimation (lyophilization).…”