Abstract
Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.
Highlights
In the non-esterified form, Fatty acids (FAs) serve as precursors of hormones and yielding esters of aliphatic, cyclic and aromatic alcohols determining the structure of membrane and modulators—key players of plant
We have shown that Langmuir–Blodgett films can be efficiently implemented in selective enrichment of phosphopeptides [22], protein adducts of organophosphorous compounds [23] and chlorinated insecticides [24], and proved to be an efficient tool for high-throughput and sensitive fingerprinting of free fatty acids as their barium monocarboxylates in positive ion mode [25]
The analyses revealed nine fatty acid signals in total, which were annotated in the both species by their exact m/z values with a mass accuracy better than 10 ppm (Table 1), i.e., within the instrument specifications
Summary
Yielding of aliphatic, cyclic and aromatic alcohols determining structure of membrane and onesters one hand, these compounds are readily involved in a widethe array of enzymatic reactions storage lipids [2]. In the non-esterified form, FAs serve as precursors of hormones and yielding esters of aliphatic, cyclic and aromatic alcohols determining the structure of membrane and modulators—key players of plant [3] non-esterified and animal [4]form, regulatory pathways, involved in all vital storage lipids [2]. Not less important the role free fattypathways, acids (FFAs) as lowinmolecular and modulators—key players of plant [3] andisanimal [4]of regulatory involved all vital weight effectors, directly in immune all kingdoms living organisms [4].
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