TY - JOUR

T1 - High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

AU - Gladchuk, Aleksey

AU - Shumilina, Julia

AU - Kusnetsova , Alena

AU - Bureiko , Ksenia

AU - Billig, Susan

AU - Tsarev , Alexander

AU - Alexandrova , Irina

AU - Leonova, Larisa

AU - Zhukov, Vladimir A.

AU - Tikhonovich , Igor A.

AU - Birkemeyer, Claudia

AU - Podolskaya, Ekaterina

AU - Frolov, Andrej

N1 - Publisher Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2020/6

Y1 - 2020/6

N2 - 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.

AB - 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.

KW - Bacteria

KW - barium monocarboxylates

KW - chemical deposition technique

KW - free fatty acids (FFAs)

KW - Langmuir film technology

KW - matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS)

KW - metabolic fingerprinting

KW - rhizobia

KW - Barium monocarboxylates

KW - Rhizobia

KW - Free fatty acids (FFAs)

KW - Metabolic fingerprinting

KW - Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS)

KW - Chemical deposition technique

UR - https://www.mdpi.com/2409-9279/3/2/36

UR - http://www.scopus.com/inward/record.url?scp=85089848948&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/5ee4ab8f-f23b-35e0-b652-90156208fad0/

U2 - 10.3390/mps3020036

DO - 10.3390/mps3020036

M3 - Article

VL - 3

SP - 1

EP - 17

JO - Methods and Protocols — Open Access Journal

JF - Methods and Protocols — Open Access Journal

SN - 2409-9279

IS - 2

M1 - 36

ER -