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.
Highlights
IntroductionMetabonomics studies, which can be used for biomarker identification for early disease detection and drug development, are accomplished by examining the differences in metabolic profiles of biological fluids between control samples and samples obtained under some form of transformative change or intrinsic difference such as the presence, progression or treatment of disease [2,3,4,5]
Metabonomics is a metabolic profiling technique first described in 1999 [1]
The absence of trimethylamine in the lyophilized samples could be due to Representative 1D nuclear magnetic resonance spectroscopy (NMR) spectra of samples prepared from redissolved speed vacuum dried and chemical conversion during speed-vacuum drying that takes place at a higher temperature compared lyophilized Panc-1 cell extracts used for metabolic profiling analysis are shown in Figure 1 with a to during lyophilization
Summary
Metabonomics studies, which can be used for biomarker identification for early disease detection and drug development, are accomplished by examining the differences in metabolic profiles of biological fluids between control samples and samples obtained under some form of transformative change or intrinsic difference such as the presence, progression or treatment of disease [2,3,4,5]. Metabonomics has been employed in food science to assess food quality and in nutrition studies to identify diet–health relationships by examination of dietary biomarkers [6,7,8,9,10], in fields such as toxicology and immunology to understand the mechanisms of chemical toxicity and viral infections [11,12], to determine how the use of oral antibiotics alters the gut microbiome based on analysis of fecal extracts [13,14], and to assess the consequences of different forms of acute kidney injury [15,16,17], among many other applications.
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