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High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

1
Institute of Toxicology, Federal Medical-Biological Agency of Russia, 192019 Saint Petersburg, Russia
2
Department of Biochemistry, St. Petersburg State University, 199034 Saint Petersburg, Russia
3
Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, 06120 Halle, Germany
4
Institute of Analytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, 04103 Leipzig, Germany
5
All-Russia Research Institute for Agricultural Microbiology, 196608 Saint Petersburg, Russia
6
Department of Genetics and Biotechnology, St. Petersburg State University, 199034 Saint Petersburg, Russia
7
Institute of Analytical Instrumentation, Russian Academy of Sciences, 198095 Saint Petersburg, Russia
*
Author to whom correspondence should be addressed.
These authors contributed equally to the manuscript.
Methods Protoc. 2020, 3(2), 36; https://doi.org/10.3390/mps3020036
Received: 13 January 2020 / Revised: 29 April 2020 / Accepted: 29 April 2020 / Published: 4 May 2020
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. View Full-Text
Keywords: bacteria; barium monocarboxylates; chemical deposition technique; free fatty acids (FFAs); Langmuir film technology; matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS); metabolic fingerprinting; rhizobia bacteria; barium monocarboxylates; chemical deposition technique; free fatty acids (FFAs); Langmuir film technology; matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS); metabolic fingerprinting; rhizobia
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Gladchuk, A.; Shumilina, J.; Kusnetsova, A.; Bureiko, K.; Billig, S.; Tsarev, A.; Alexandrova, I.; Leonova, L.; Zhukov, V.A.; Tikhonovich, I.A.; Birkemeyer, C.; Podolskaya, E.; Frolov, A. High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Methods Protoc. 2020, 3, 36.

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