The Importance of Sample Preparation for Omics Analysis: Which Extraction Method is the most Suited for my Biological Question?

The instrumental aspect employed in metabolomic and lipidomic strategies has been well developed as of the past two decades, allowing large-scale analysis with high sensitivity and reproducibility, while statistical analysis and bioinformatics are emerging fields for data interpretation. Conversely, sample preparation is an important element that, if not optimized, has an impact on metabolome profiling and may result in erroneous and biased biological interpretation, remaining the bottleneck of the metabolomics workflow. Therefore, this work presents an evaluation of liquid-liquid extraction protocols for blood serum samples aiming at metabolomics and lipidomics analysis using liquid chromatography coupled to mass spectrometry (LC-MS). The Bligh & Dyer, Matyash and SIMPLEX protocols were investigated and the findings demonstrate that the composition of the solvents in each approach has a considerable impact on the putatively identified lipids, as well as the average concentration of extracted proteins. The study of the aqueous fraction, however, revealed no statistically significant variations in the identified metabolites. Hence, the optimization of the sample preparation step is crucial for biomolecules profiling and must be carefully considered in accordance with the objectives of the research and the chosen biological matrices.