Quantification of phosphorylated metabolites, organic acids, and intermediates of the TCA cycle using capillary ion chromatography tandem mass spectrometry (capIC-MS/MS) following treatment of Escherichia coli with ciprofloxacin

Data for the following article published in Frontiers in Microbiology: Bergum OET, Singleton AH, Røst LM, Bodein A, Scott-Boyer M-P, Rye MB, Droit A, Bruheim P and Otterlei M (2024) SOS genes are rapidly induced while translesion synthesis polymerase activity is temporally regulated. Front. Microbiol. 15:1373344. doi: 10.3389/fmicb.2024.1373344 Capillary ion chromatography tandem mass spectrometry (capIC-MS/MS) was used to quantify phosphorylated metabolites, organic acids, and intermediates of the TCA cycle of Escherichia coli treated with ciprofloxacin, BTP-001 (a novel antimicrobial peptide), and a combination of the two . Metabolite extracts were analyzed with a Xevo TQ-XS triple quadrupole mass spectrometer (Waters, USA). Samples were gathered from E. coli cultures grown in batch cultivations using 1 liter bioreactors. Briefly, intracellular metabolites were extracted by cycling samples between −20 °C EtOH and N2 (l) in three consecutive freeze–thaw cycles, with vortexing every 10 min during the thawing phase. Filters were removed and the cell debris was pelleted (4500 rcf, 10 min, -9 °C). The supernatants were transferred to a new tube, snap frozen in N2 (l), and lyophilized. Lyophilized extracts were reconstituted in 500 µL cold Milli-Q H2O and cleared by spin-filtration with a 10 kDa molecular cutoff (20817 rcf, 10 min, 0 °C). A mix of 80 µL centrifuged sample and 20 µL 13C-labeled ISTD extract from yeast was sent to analysis. Data processing and absolute quantification was performed as earlier described using the TargetLynx application manager of MassLynx v 4.1 (Waters) to interpolate calibration curves made with appropriate dilutions of analytical grade standards (Sigma-Aldrich). The response factor of the corresponding U13C-isotopologues were used to correct the standard and sample extract response factors. Extract concentrations were normalized to the CDW, which was calculated from interpolation of the OD600 vs. CDW (g/L) curve. Further statistical analysis in MetaboAnalyst v 5.0 replaced missing values with 1/5 of the minimum value of the respective metabolite. An unpaired T-test with unequal variance determined differential enriched metabolites with a false discovery rate (FDR) < 0.05 which are presented as log2 fold-change compared to control.本数据集对应发表于《微生物学前沿》（Frontiers in Microbiology）的以下论文： Bergum OET、Singleton AH、Røst LM、Bodein A、Scott-Boyer M-P、Rye MB、Droit A、Bruheim P及Otterlei M（2024）。论文标题为《SOS基因在跨损伤合成聚合酶活性受时序调控时被快速诱导》，发表于Front. Microbiol. 15:1373344，DOI: 10.3389/fmicb.2024.1373344。 采用毛细管离子色谱-串联质谱（capIC-MS/MS）对经环丙沙星、BTP-001（一种新型抗菌肽）及二者联合处理的大肠杆菌的磷酸化代谢物、有机酸以及三羧酸（TCA）循环中间产物进行定量分析。代谢提取物使用Xevo TQ-XS三重四极杆质谱仪（美国沃特世（Waters）公司）进行检测。 样本取自采用1升生物反应器进行分批培养的大肠杆菌培养液。简言之，细胞内代谢物通过将样本在-20℃乙醇与液氮（N₂(l)）之间循环进行3次连续冻融循环提取，解冻阶段每10分钟进行一次涡旋混匀。移除滤膜后，将细胞碎片离心沉淀（4500相对离心力，10分钟，-9℃）。将上清液转移至新管，经液氮快速冷冻后进行冻干处理。将冻干提取物用500 μL预冷的Milli-Q级超纯水复溶，随后采用10 kDa分子量截留膜进行离心过滤净化（20817相对离心力，10分钟，0℃）。取80 μL离心后的样本与20 μL酵母来源的13C标记内标（ISTD）提取物混合后进行分析。 数据处理与绝对定量方法参照此前报道，使用MassLynx v4.1软件（沃特世）的TargetLynx应用管理器，通过对分析级标准品（西格玛奥德里奇（Sigma-Aldrich））的系列稀释液绘制校准曲线并进行插值计算。采用对应U13C同位素异构体的响应因子对标准品与样本提取物的响应因子进行校正。提取物浓度以细胞干重（CDW）进行归一化处理，细胞干重通过OD600与细胞干重（g/L）标准曲线插值计算得到。 使用MetaboAnalyst v5.0进行进一步统计分析时，将缺失值替换为对应代谢物最小值的1/5。采用方差不齐的非配对t检验筛选差异富集代谢物，以错误发现率（FDR）<0.05作为筛选阈值，结果以与对照组相比的log2倍数变化值呈现。