Gladchuk Aleksey, Shumilina Julia, Kusnetsova Alena, Bureiko Ksenia, Billig Susan, Tsarev Alexander, Alexandrova Irina, Leonova Larisa, Zhukov Vladimir A, Tikhonovich Igor A, Birkemeyer Claudia, Podolskaya Ekaterina, Frolov Andrej
Institute of Toxicology, Federal Medical-Biological Agency of Russia, 192019 Saint Petersburg, Russia.
Department of Biochemistry, St. Petersburg State University, 199034 Saint Petersburg, Russia.
Methods Protoc. 2020 May 4;3(2):36. doi: 10.3390/mps3020036.
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 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- and , demonstrates the analytical potential of the protocol.
脂肪酸（FAs）是一类重要的代谢产物，对细胞调节网络中的膜结构单元和信号化合物产生影响。在自然界中，原核生物具有最令人印象深刻的脂肪酸结构多样性和最高的游离脂肪酸（FFAs）相对含量。在这种背景下，作为豆科植物共生体的固氮细菌（根瘤菌目）尤其引人关注。事实上，脂肪酸谱会影响根瘤菌结瘤因子的结构，而结瘤因子是成功感染植物根系所必需的。虽然脂肪酸模式可以通过气相色谱 -（GC-）和液相色谱 - 质谱联用（LC-MS）进行评估，但这些方法的样品制备耗时，且定量存在灵敏度受损、衍生物稳定性低和假象等问题。相比之下，基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）是一种用于高效代谢物指纹识别的优秀平台，也适用于游离脂肪酸。因此，在这里我们提出一种简单直接的方案，通过结合朗缪尔技术和MALDI-TOF-MS对根瘤菌中的游离脂肪酸进行高通量相对定量，该方案具有高灵敏度、准确性和定量精度。我们描述了一个包括根瘤菌培养、预清洁、提取、样品制备、质谱分析、数据处理和后处理的程序。作为一个案例研究，对两种根瘤菌的游离脂肪酸代谢组进行比较，证明了该方案的分析潜力。
