1 Ocak 2022 Dergi makalesi Açık Erişim

Eylem, Cemil Can; Nemutlu, Emirhan; Dogan, Aysegul; Acik, Vedat; Matyar, Selcuk; Gezercan, Yurdal; Altintas, Suleyman; Okten, Ali Ihsan; Akduman, Nursabah Elif Basci

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    <subfield code="a">To acquire deeper insights into the underlying molecular mechanisms in biological systems, a comprehensive and in-depth examination of a wide diversity of molecular species is necessary. Especially, it is very crucial to examine intermediary molecular levels such as protein, metabolite, and lipid to demonstrate direct causative and functional linkages between genotype and phenotype. Therefore, multiple sample sets/aliquots and analytical methods are required to cover different intermediary molecular levels due to the dissimilarity of physicochemical characteristics of biomolecules. However, existing methods used for simultaneous analysis of metabolites and lipids have not been thoroughly tested for reproducibility and wide applicability. Here, we have developed and optimized high-throughput and robust metabolomics and lipidomics analysis using experimental design strate-gies for single-step extraction protocols and also method parameters. Due to the high number of variables, an approach based on a desirability function was applied to optimize sample preparation and chromatographic parameters of metabolomics and lipidomics analysis. In these experiments, metabolite extraction efficiency was tested with acetone, acetonitrile, ethanol, methanol, water, and their combination, while lipids with hexane, chloroform, dichloromethane, and methyl tert-butyl ether. In the presented study, it is found that methanol: water:chloroform (3:1:3, v/v/v) mixture was superior to the other extraction solvent combinations in all per-formance measures. Moreover, the derivatization steps in GC-MS and chromatographic parameters in LC-qTOF-MS were tested. The derivatization efficiency increased with a higher concentration of methoxyamine hydro-chloride and incubation time with the derivatization reagent. For LC-qTOF-MS, reconstitution solution, and the column temperature was found critical for high throughput metabolomics and lipidomics analysis, respectively. This systematic optimization for sample preparation and method parameters for integrated GC-MS and LC-qTOF-MS allows simultaneous and high-throughput analysis of metabolites as well as lipids from a single sample, making it a simple and practical strategy for different sample types.</subfield>
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