Analyzing proteins and metabolites: All-in-One Extraction
Optimized analysis of selected metabolites or classes of metabolites like polar amino acids or more unipolar triacylglycerols (TAG), requires specific extraction procedures in combination with specific analytical instrumentation. However, the most efficient, specific extraction protocols unfortunately often only cover a very limited number of compounds.
As a consequence, many samples are needed to reach a comprehensive metabolic coverage. For many plants, the amount of starting material might not be a limitation, but time- and effort-wise a single extraction procedure, covering most of the metabolic classes, would be a preferred solution to minimize the time for sample preparation and analysis and to maximize the throughput. For this purpose sequential or fractionated extraction protocols have been developed and used.
In these methods, several metabolic classes are extracted iterative by increasing hydrophobicity of the extraction buffer or by using a complex extraction buffer, containing solvents of different polarity. The classical approach is the methanol:chloroform:water extraction protocol, where several different metabolite classes are separated in a liquid:liquid separation step. The polar phase, of this extraction protocol contains polar primary- and semi-polar secondary metabolites, which can be analyzed by GC- and LC-MS-based metabolomic analysis. The lipid-containing organic (chloroform) phase could be analyzed by direct-infusion-based MS/MS or by UPLC-MS.
In the methanol:chloroform extraction proteins and starch are hardly accessible
Even though the Methanol:Chloroform:Water extraction is quite efficient for the metabolite extractions, we found that it had a major limitation for the systematic analysis of proteins, next to the metabolites. The problem in the methanol:chloroform extraction was, that proteins and starch sediment to the diffuse interphase, between the lower organic and the upper polar phase, which makes these compounds hardly accessible. To overcome this problem, we sought for an alternative organic solvent, substituting the heavy chloroform, which was forming the protein impermeable lower phase of our extraction procedure, by a less dense solvent.
Extraction with MTBE gives rise to three independent fractions
Interestingly, we found that methyl-tertiary-butyl-ether (MTBE) was used as a substitute for chloroform in lipidomic studies, which perfectly matched our needs. The newly developed extraction procedure, similar to the previous chloroform-containing method, led to a liquid:liquid separation, but now with an inverted phases. The lipid containing MTBE phase is now on top of the heavier methanol/water phase. This enabled us to harvest the starch and proteins from a solid pellet in the bottom of the Eppendorf tube. Taken together, our optimized extraction protocol allows now to extract three independent fractions, of which the protein/starch pellet can be directly used for proteomic analysis (see later section), while the lower polar phase from the liquid:liquid system can be used for GC-MS-based analysis of primary metabolites and LC-MS-based analysis of semi-polar secondary metabolites. Finally the upper organic phase can be directly used for lipidomic analysis.