Cellular metabolomics of Escherchia coli

Escherichia coli is among the simplest and best-understood free-living organisms. It has served as a valuable model for numerous biological processes, including cellular metabolism. Just as E. coli stood at the front of the genomic revolution, it is playing a leading role in the development of cellular metabolomics: the study of the complete metabolic contents of cells, including their dynamic concentration changes and fluxes. This review briefly describes the essentials of cellular metabolomics and its fundamental differentiation from biomarker metabolomics and lipidomics. Key technologies for metabolite quantitation from E. coli are described, with a focus on those involving mass spectrometry. In particular emphasis is given to the cell handling and sample preparation steps required for collecting data of high biological reliability, such as fast metabolome quenching. Future challenges, both in terms of data collection and application of the data to obtain a comprehensive understanding of metabolic dynamics, are discussed.     

Cold glycerol-saline: the promising quenching solution for accurate intracellular metabolite analysis of microbial cells

Microbial metabolomics has been seriously limited by our inability to perform a reliable separation of intra- and extracellular metabolites with efficient quenching of cell metabolism. Microbial cells are sensitive to most (if not all) quenching agents developed to date, resulting in leakage of intracellular metabolites to the extracellular medium during quenching. Therefore, as yet we are unable to obtain an accurate concentration of intracellular metabolites from microbial cell cultures. However, knowledge of the in vivo concentrations of intermediary metabolites is of fundamental importance for the characterization of microbial metabolism so as to integrate meaningful metabolomics data with other levels of functional genomics analysis. In this article, we report a novel and robust quenching method for microbial cell cultures based on cold glycerol-saline solution as the quenching agent that prevents significant leakage of intracellular metabolites and, therefore, permits more accurate measurement of intracellular metabolite concentrations in microbial cells.     

Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells

Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus–insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl + P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell–baculovirus systems for bioprocess optimization.     

Global metabolomics characterization of bacteria: pre-analytical treatments and profiling

Pre-analytical treatments of bacteria are crucial steps in bacterial metabolomics studies. In order to achieve reliable samples that can best represent the global metabolic profile in vivo both qualitatively and quantitatively, many sample treatment procedures have been developed. The use of different methods makes it difficult to compare the results among different groups. In this work, E. coli samples were tested by using NMR spectroscopy. Both liquid N₂ and cold methanol quenching procedures reduce the cell membrane integrity and cause metabolites leakage. However, liquid N₂ quenching affected the cell viability and the NMR metabolites’ profile less than cold methanol procedure. Samples obtained by metabolite extraction were significantly superior over cell suspensions and cell lysates, with a higher number of detectable metabolites. Methanol/chloroform extraction proved most efficient at extraction of intracellular metabolites from both qualitative and quantitative points of view. Finally, standard operating procedures of bacterial sample treatments for NMR metabolomics study are presented.     

Optimizing high-throughput metabolomic biomarker screening: a study of quenching solutions to freeze intracellular metabolism in CHO cells

Metabolomics is a rapidly emerging tool for studying and optimizing both media and bioprocess development for culturing recombinant mammalian cells that are used in protein production processes. Quenching of the cells is crucial to fix their metabolic status at the time of sampling. Three precooled quenching solutions were tested for their ability to fix the metabolic activity of CHO cells: phosphate-buffered saline (PBS) (pH 7.4; 0.5°C), 60% methanol with 70?mM HEPES (pH 7.4; -20°C), and 60% methanol with 0.85% (w/v) ammonium bicarbonate (AMBIC) (pH 7.4; -20°C). The metabolic activity of the sampled CHO cells was assessed by determining the intracellular levels of ATP using a bioluminescence assay and selected metabolites with LC-MS/MS. We found the precooled PBS (pH 7.4; 0.5°C) to be the optimal quenching reagent for fixing intracellular metabolism. Importantly, the structural integrity of the cell membrane was maintained and highest yields were obtained for intracellular levels of ATP as well as for 18 out of 28 intracellular metabolites. In contrast to the previously reported studies, buffered methanol quenching was not applicable for suspension cultured CHO cells as cellular membrane integrity was affected. We recommend that the cells are quenched and washed simultaneously to keep the sampling time to a minimum and to prevent any further metabolic activity in the cells. We observed that additional washing steps are not required. Our analyses suggest that methanol as quenching solution, even in combination with a buffer substance, appears not suitable for quenching sensitive mammalian cells. The protocol we report herein is a simple cell sampling method that enables high-throughput metabolomic analyses and is suitable for a large number of samples.     

植物代谢组学中几种重要的次生代谢物液质分析技术研究进展

代谢组学（metabolomics）主要是研究生物体、组织、细胞的代谢物组分及检测其动态变化过程,是继基因组和蛋白组学后新兴的一门组学技术。代谢物是细胞调节过程中的最终产物,其水平被视为生物系统对遗传或环境变化的最终反映。通过合适的分析平台,准确定性、定量在复杂的生物中具有化学多样性的次生代谢物是代谢组学的一项重要工作。液相色谱-串联质谱技术（liquid chromatography-tandem mass spectrometry,LC-MS/MS）是代谢物质检测平台最常用的方法,也为植物次生代谢物的广泛应用研究提供了基础。本文主要从植物激素类、叶酸类、黄酮类等次生代谢物方面进行阐述,结合液质联用技术,简要论述不同次生代谢物检测技术的研究进展。     

苜蓿中华根瘤菌代谢组学样品制备方法的研究

代谢组样品制备是代谢组学研究的基础。本文以维生素B12生产菌株苜蓿中华根瘤菌Sinorhizobium meliloti 320为研究对象,通过检测细胞损伤、ATP泄漏、代谢物回收效率以及细胞代谢淬灭效率综合评价细胞淬灭方法,同时对5种提取试剂的提取效率进行比较优化胞内代谢物的提取方法。最终获得苜蓿中华根瘤菌S.meliloti 320的胞内代谢组学样品制备较佳条件:即-20℃40%甲醇淬灭细胞,过滤收集淬灭细胞,甲醇/乙腈/水(体积比为2∶2∶2,外加0.1%的甲酸)与50%甲醇相结合提取胞内代谢物。实验结果显示-20℃的40%甲醇(通过过滤收集细胞)对细胞膜的损伤较小,且细胞代谢淬灭效率和回收效率较高;甲醇/乙腈/水(体积比为2∶2∶2,外加0.1%的甲酸)与50%的甲醇对胞内代谢物的提取效率较高且有互补作用。     

微生物代谢组学及其在工业中的应用

微生物代谢组学是系统生物学的重要组成部分,其与基因组学、转录组学和蛋白质组学相互补充,近年来受到越来越多人的重视。其主要对细胞生长或生长周期某一时刻细胞内外所有低分子量代谢物进行定性和定量分析,直接反映了细胞的生理状态,对理解细胞功能十分重要。由于代谢物的复杂性,研究者需根据不同的目的及对象选择合适的分析方法。对微生物代谢组学近年来的研究方法进行综述,包括样品处理、分析手段、数据分析,并讨论了微生物代谢组学在工业中的应用及所面临的挑战。     

The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

Metabolism is vital to every aspect of cell function, yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis, and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly, the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs, which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs, and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency, and in evaluating iPSC and ESC equivalence.     

Microbial metabolomics: past,present and future methodologies

Microbial metabolomics has received much attention in recent years mainly because it supports and complements a wide range of microbial research areas from new drug discovery efforts to metabolic engineering. Broadly, the term metabolomics refers to the comprehensive (qualitative and quantitative) analysis of the complete set of all low molecular weight metabolites present in and around growing cells at a given time during their growth or production cycle. This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites as well as quantification of these metabolites using enzyme based and or modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (LC-MSⁿ, GC-MSⁿ, CE-MSⁿ), where n indicates the number of tandem mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR).     

Automated fast filtration and on‐filter quenching improve the intracellular metabolite analysis of microorganisms

To reliably determine intracellular metabolite concentrations in microorganisms, accurate sampling and sample inactivation strategies are crucial. Here, we present a method for automated fast filtration and on‐filter quenching of microbial samples to overcome metabolite leakage induced by cold shock and significantly reduce the sampling and treatment time compared to manual filtration methods. The whole process of sampling, sample filtration, filter wash, and quenching of the filter with liquid nitrogen was finished in less than 6–15 s, depending on the experimental setup. By integration into an automated fast sampling device, we compared our method to the conventional methanol quenching method and showed that intracellular amino acid contents in Escherichia coli were significantly increased (≥75%) with our fast filtration and on‐filter quenching method. Furthermore, we investigated different filter types for the fast filtration and the efficiency of metabolite extraction from cells held on filters. Additionally, we found that the fast filtration behaves considerably different during exponential and nonexponential growth, probably due to variations of cell morphologies. Overall, we demonstrated that the automation of the fast filtration method significantly reduces the time for filtration and quenching and hence enlarge the number of metabolites that can be quantified with this leakage‐free sampling method.     

An improved sampling protocol for analysis of intracellular metabolites in Mortierella alpina

Sampling of intracellular metabolites in Mortierella alpina was investigated as part of a metabolomics study. After comparison of four sampling protocols, rapid filtration of the culture using a laboratory-made nylon filter and absorbent gauze under normal pressure followed by quenching in liquid N₂ and grinding (the improved protocol) was the most effective. Rapid filtration under normal pressure decreased intracellular metabolites leakage and subsequent grinding of cells contributed to intracellular metabolites extraction. The above quenching method together with 75?% (v/v) ethanol, buffered with 60?mM HEPES, at 80?°C for 3?min is therefore suitable for sampling intracellular metabolites in M. alpina.     

紫杉二烯人工酵母的代谢组学分析

为了更深入地从代谢角度研究萜类合成人工酵母的内在差异,以紫杉二烯人工酵母为例,利用代谢组学的方法对其发酵指数中期胞内代谢物的变化进行了测定。结果表明,与对照菌W303-1A相比,紫杉二烯的生产会对胞内糖酵解、三羧酸循环中间物及一些氨基酸的含量产生不同程度的影响,进而对其生长产生一定抑制作用。其中柠檬酸因紫杉二烯功能模块的引入下降明显,降幅达90%以上,因此可以作为后续功能酵母研究的标志性代谢物。紫杉二烯人工酵母细胞代谢组的研究可以为萜类化合物异源合成的优化提供更多的信息和帮助。     

LC-MS metabolic profiling of Arabidopsis thaliana plant leaves and cell cultures: optimization of pre-LC-MS procedure parameters

This study treats the optimization of methods for homogenizing Arabidopsis thaliana plant leaves as well as cell cultures, and extracting their metabolites for metabolomics analysis by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). Absolute recovery, process efficiency and procedure repeatability have been compared between different pre-LC-MS homogenization/extraction procedures through the use of samples fortified before extraction with a range of representative metabolites. Hereby, the magnitude of the matrix effect observed in the ensuing LC-MS based metabolomics analysis was evaluated. Based on relative recovery and repeatability of key metabolites, comprehensiveness of extraction (number of m/z-retention time pairs) and clean-up potential of the approach (minimum matrix effects), the most appropriate sample pre-treatment was adopted. It combines liquid nitrogen homogenization for plant leaves with thermomixer based extraction using MeOH/H(2)O 80/20. As such, an efficient and highly reproducible LC-MS plant metabolomics set-up is achieved, as illustrated by the obtained results for both LC-MS (8.88%+/-5.16 versus 7.05%+/-4.45) and technical variability (12.53%+/-11.21 versus 9.31%+/-6.65) data in a comparative investigation of A. thaliana plant leaves and cell cultures, respectively.     

Leakage-free rapid quenching technique for yeast metabolomics

Accurate determination of intracellular metabolite levels requires reliable, reproducible techniques for sampling and sample treatment. Quenching in 60% (v/v) methanol at −40°C is currently the standard method for sub-second arrest of metabolic activity in microbial metabolomics but there have been contradictory reports in the literature on whether leakage of metabolites from the cells occurs. We have re-evaluated this method in S. cerevisiae using a comprehensive, strictly quantitative approach. By determining the levels of a large range of metabolites in different sample fractions and establishing mass balances we could trace their fate during the quenching procedure and confirm that leakage of metabolites from yeast cells does occur during conventional cold methanol quenching, to such an extent that the levels of most metabolites have been previously underestimated by at least twofold. In addition, we found that the extent of leakage depends on the time of exposure, the temperature and the properties of the methanol solutions. Using the mass balance approach we could study the effect of different quenching conditions and demonstrate that leakage can be entirely prevented by quenching in pure methanol at ≤−40°C, which we propose as a new improved method. Making use of improved data on intracellular metabolite levels we also re-evaluated the need of sub-second quenching of metabolic activity and of removing the extracellular medium. Our findings have serious implications for quantitative metabolomics-based fields such as non-stationary ¹³C flux analysis, in vivo kinetic modeling and thermodynamic network analysis.

Metabolomics assisted metabolic network modeling and network wide analysis of metabolites in microbiology

Metabolomics is the science of qualitatively and quantitatively analyzing low molecular weight metabolites occur in a given biological system. It provides valuable information to elucidate the functional roles and relations of different metabolites in a metabolic pathway. In recent years, a large amount of research on microbial metabolomics has been conducted. It has become a useful tool for achieving highly efficient synthesis of target metabolites. At the same time, many studies have been conducted over the years in order to integrate metabolomics data into metabolic network modeling, which has yielded many exciting results. Additionally, metabolomics also shows great advantages in analyzing the relationship of metabolites network wide. Integrating metabolomics data into metabolic network construction and applying it in network wide analysis of cell metabolism would further improve our ability to control cellular metabolism and optimize the design of cell factories for the overproduction of valuable biochemicals. This review will examine recent progress in the application of metabolomics approaches in metabolic network modeling and network wide analysis of microbial cell metabolism.     

Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling

Metabolite profiling of industrially important suspension-cultured mammalian cells is being increasingly used for rational improvement of bioprocesses. This requires the generation of global metabolite profiles that cover a broad range of metabolites and that are representative of the cells at the time of sampling. The protocol described here is a validated method for recovery of physiologically relevant amounts of key metabolites from suspension-cultured mammalian cells. The method is a two-step process consisting of initial quenching of the cells (to stop cellular metabolism and allow isolation of the cells) followed by extraction of the metabolites. The cells are quenched in 60% methanol supplemented with 0.85% (wt/vol) ammonium bicarbonate at -40 °C. Metabolites are then extracted from the quenched cells using two 100% methanol extractions followed by a single water extraction. Metabolite samples generated using this protocol are amenable to analysis by mass spectrometry-based techniques (e.g., gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry), NMR spectroscopy and enzymatic assays.     

Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum

Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra‐ and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration‐based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration‐based washing. In the centrifugation‐based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin‐G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3–4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin‐G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood. Biotechnol. Bioeng. 2010;107: 105–115. © 2010 Wiley Periodicals, Inc.     

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

Quantitative metabolomics of microbial cultures requires well-designed sampling and quenching procedures. We successfully developed and applied a differential method to obtain a reliable set of metabolome data for Escherichia coli K12 MG1655 grown in steady-state, aerobic, glucose-limited chemostat cultures. From a rigorous analysis of the commonly applied quenching procedure based on cold aqueous methanol, it was concluded that it was not applicable because of release of a major part of the metabolites from the cells. No positive effect of buffering or increasing the ionic strength of the quenching solution was observed. Application of a differential method in principle requires metabolite measurements in total broth and filtrate for each measurement. Different methods for sampling of culture filtrate were examined, and it was found that direct filtration without cooling of the sample was the most appropriate. Analysis of culture filtrates revealed that most of the central metabolites and amino acids were present in significant amounts outside the cells. Because the turnover time of the pools of extracellular metabolites is much larger than that of the intracellular pools, the differential method should also be applicable to short-term pulse response experiments without requiring measurement of metabolites in the supernatant during the dynamic period.     

胎鼠脊髓神经干细胞分离方法的比较

目的：比较机械法和胰酶消化法对胎鼠脊髓源神经干细胞增殖分化的影响。方法：分别用机械法和胰酶消化法分离胎鼠脊髓组织获得神经干细胞,应用台盼蓝检测细胞成活率,用无血清培养技术培养神经干细胞,应用MTT法检测细胞分裂增殖能力．采用免疫细胞化学法鉴定神经干细胞和分化细胞。结果：机械法获得的细胞数量多于胰酶消化法。细胞经过培养其增殖能力机械法略强于胰酶消化法,但无统计学意义。培养形成的细胞球Nestin阳性,诱导分化后可见NSE和GFAP阳性细胞。结论：运用机械法比胰酶消化法分离胎鼠脊髓组织获得神经干细胞方法简单,容易操作,经过培养细胞增殖能力较强。并可提供健康的细胞来源。