Protocol: a fast, comprehensive and reproducible one-step extraction method for the rapid preparation of polar and semi-polar metabolites, lipids, proteins, starch and cell wall polymers from a single sample

Abstract

BackgroundThe elucidation of complex biological systems requires integration of multiple molecular parameters. Accordingly, high throughput methods like transcriptomics, proteomics, metabolomics and lipidomics have emerged to provide the tools for successful system-wide investigations. Unfortunately, optimized analysis of different compounds requires specific extraction procedures in combination with specific analytical instrumentation. However, the most efficient extraction protocols often only cover a restricted number of compounds due to the different physico-chemical properties of these biological compounds. Consequently, comprehensive analysis of several molecular components like polar primary metabolites next to lipids or proteins require multiple aliquots to enable the specific extraction procedures required to cover these diverse compound classes. This multi-parallel sample handling of different sample aliquots is therefore not only more sample intensive, it also requires more time and effort to obtain the required extracts.ResultsTo circumvent large sample amounts, distributed into several aliquots for the comprehensive extraction of most relevant biological compounds, we developed a simple, robust and reproducible two-phase liquid–liquid extraction protocol. This one-step extraction protocol allows for the analysis of polar-, semi-polar and hydrophobic metabolites, next to insoluble or precipitated compounds, including proteins, starch and plant cell wall components, from a single sample. The method is scalable regarding the used sample amounts but also the employed volumes and can be performed in microcentrifuge tubes, enabling high throughput analysis. The obtained fractions are fully compatible with common analytical methods, including spectroscopic, chromatographic and mass spectrometry-based techniques. To document the utility of the described protocol, we used 25 mg of Arabidopsis thaliana rosette leaves for the generation of multi-omics data sets, covering lipidomics, metabolomics and proteomics. The obtained data allowed us to measure and annotate more than 200 lipid compounds, 100 primary metabolites, 50 secondary metabolites and 2000 proteins.ConclusionsThe described extraction protocol provides a simple and straightforward method for the efficient extraction of lipids, metabolites and proteins from minute amounts of a single sample, enabling the targeted but also untargeted high-throughput analyses of diverse biological tissues and samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0146-2) contains supplementary material, which is available to authorized users.

Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii

“…In the current workflow Chlamydomonas cells were harvested by centrifugation, but the protocol can also be used with any other strategy for the fast and reproducible sample collection (Huege et al ., ; Hemme et al ., ), especially if labile metabolites like sugar phosphates are to be measured (Arrivault et al ., ). The recently updated version of the MTBE method allowed for the simultaneous extraction of chlorophyll, proteins, starch, polar and semi‐polar metabolites, lipids and cell wall components from 25 μg of plant tissue (Salem et al ., ).…”

“…A prerequisite for the detailed, systemic analysis of a biological sample is a robust and high‐throughput‐compatible extraction method for multiple cellular components. We used a previously developed plant tissue extraction method (Salem et al ., ) for the extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of Chlamydomonas cell cultures (Figure S6). The use of such an extraction method offers several advantages for multiple‐omics data analysis, since it not only minimizes the number of sample aliquots required for broad systems biological analyses but also reduces the variability between the analytical results.…”

MS-CleanR: A feature-filtering approach to improve annotation rate in untargeted LC-MS based metabolomics

“…The RMD is calculated in ppm as [(mass defect/measured monoisotopic mass) × 10 6 )]. It can be used to filter compound classes 23 and it should also be useful to remove artifactual signals. Based on all compounds exported from the Dictionary of Natural Products (DNP; available on DVD v.28.2 from CRC Press), we found that 95% of natural products (NP)s had RMD values of 156.5-969.6 ppm.…”

“…Biphasic sample extraction was adapted from Salem et al . 2016 23 by adding two cycles of 20 seconds at 6 m/sec. in the FastPrep-24™ benchtop homogenizer (MP Biomedicals™, Illkirch, France) in 1 mL M1 (methyl tert-butyl ether/methanol, 3:1, v:v) extraction solution.…”

Predicting metabolism during growth by osmotic cell expansion

“…Lipids were extracted from a known mass of ground tissue using the chloroform/methanol protocol (Bligh and Dyer, 1959). Protein extracted with 6 M urea, 2 M thiourea buffer (Salem et al, 2016) was quantified using the Bradford assay (Bradford, 1976). The amino acid content of protein hydrolysates (50% w/v trichloroacetic acid, 6 M HCl, 100°C/24 h) (Antoniewicz et al, 2007) was determined by an established GC/EI-TOF-MS protocol (Luedemann et al, 2008;Fernie et al, 2011;Osorio et al, 2011).…”