微生物代谢产物检测-1 衍生化方法相关文献阅读与方法选择 2020-12-17

1.背景知识：

• 文献1-1 A rapid microwave-assisted derivatization of bacterial metabolome samples for gas chromatography/mass spectrometry analysis
  https://doi.org/10.1016/j.ab.2009.04.030
• 代谢组学基本概念：
  为了研究基因型和表型之间的联系，所谓的代谢组学，需要一种高通量的分析技术，该技术可以表征所有可想象的生物体和植物以及流体或其他与代谢物有关的基质的表型。该领域大多数实验室的主要目标是开发一种可靠，快速的方法，以从原始样品中生成全面的代谢组数据。许多研究致力于样品收集和代谢物提取，例如从细菌细胞或植物组织中提取。

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• 文献1-2
  Metabolomics – the link between genotypes and phenotypes
  O Fiehn - 《Plant Molecular Biology》DOI： 10.1007/978-94-010-0448-0_11
  代谢产物是细胞调节过程的最终产物，其水平可被视为生物系统对遗传或环境变化的最终反应。与术语“转录组”和“蛋白质组”同时使用，由生物系统合成的一组代谢产物构成其“代谢组”。

• 不同检测方法的粗略比较：
  在大多数情况下，代谢组学实验室使用液相色谱或气相色谱联用质谱检测（分别为LC / MS或GC / MS）。LC / MS方法在极少数情况下需要对分析物进行柱前或柱后衍生化，而在GC / MS分析中，由于大多数目标代谢物在其样品中都是非挥发性的，因此在对样品进行GC之前必须先进行样品衍生化原始物质。

• 通常关注的主要目标代谢物：
  主要的代谢物：有机酸，氨基酸，糖，磷酸糖，脂肪酸和类固醇，所有这些对于生物体生理都很重要。

• 衍生化的目的与结果：
  在大多数方法中，极性基团和活泼氢原子（例如：–OH，NH，–COOH，–SH和其他化学基团）都用N-甲基-N-三甲基甲硅烷基三氟乙酰胺（MSTFA）或N，O-·双三甲基甲硅烷基三氟乙酰胺（BSTFA）进行衍生化，从而获得更多挥发性化合物。通过在甲硅烷基化之前向样品中加入甲氧基胺（MeOx）或乙氧基胺，可以减少单糖的互变异构形式的数量，同时将醛基或酮基转化为羟胺或烷氧基胺。

• 衍生化方法与步骤
  已有文献报道许多不同的原始代谢物衍生化方法规程。其中大多数步骤需要两个步骤：首先在37°C下用乙氧基胺或MeOx进行肟化90分钟，然后在加热块中在37°C下进行三甲基硅烷化30分钟。（如果缩短代谢组学分析中耗时的预处理步骤，可以提高检测效率。）
  1）具体方法：代谢物衍生化通常用于植物，细菌和真核生物样品，方法是：将80μlMeOx（20 mg ml-1吡啶）加入冷冻干燥的样品中（即：事先需要先冻干），并在37°C加热90分钟。

2)细菌样品的处理与衍生化：
样品收集：Sampling for Metabolome Analysis of Microorganisms https://doi.org/10.1021/ac0623888
枯草芽孢杆菌菌株168和金黄色葡萄球菌COL的细菌生长条件如其他地方所述[21]。如最近发表的 [3], [17]:通过快速过滤收集细菌代谢组样品。在过滤器上洗涤细胞后，将过滤器浸入含有内标物的冰冷溶液中，以终止进一步的细胞代谢。该溶液立即在液氮中冷冻。随后用有机溶液进一步提取，并且除了金黄色葡萄球菌外，通过用玻璃珠机械破碎。离心（4°C，8000 rpm，5分钟）后，弃去细胞碎片，将上清液冻干，然后如上所述进行衍生化分析。为了捕获本构代谢物，分析了所有高于最高峰0.5％的峰.

• 文献2 Nitric oxide stress induces different responses but mediates comparable protein thiol protection in Bacillus subtilis and Staphylococcus aureus.
  https://dx.doi.org/10.1128%2FJB.01846-07

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2.合成培养基的选择：

1) Staphylococcus aureus

• 文献3 Regulation of sigmaB-dependent transcription of sigB and asp23 in two different Staphylococcus aureus strains
  S. Gertz, S. Engelmann, R. Schmid, K. Ohlsen, J. Hacker & M. Hecker

• 相关信息 (BERGEY’S MANUAL OF Systematic Bacteriology, 2nd Edition, V3, The Firmicutes)

Facultative anaerobes. With the exception of Staphylococcus aureus subsp. anaerobius and Staphylococcus saccharolyticus,growth is more rapid and abundant under aerobic conditions.
Usually catalase-positive and oxidase-negative. Most strains grow in the presence of 10% NaCl and between 18 and 40°C.

• Staphylococcus aureus strains were grown in a synthetic medium with thefollowing composition. The basic medium consisted of
  10 mM Na2HPO4,
  10mM KH2PO4,
  9.34 mM NH4Cl,
  8.55 mM NaCl,
  0.81 mM MgSO4,
  0.142 mM citric acid,
  40 mM MOPS pH 7.0,and 7.5 mM glucose.

• This was supplemented with amino acids:(L-alanine,L-arginine,L-aspartic acid,L-cysteine,L-glutamic acid,L-glycine,L-histidine,L-isoleucine,L-leucine,L-lysine,L-phenylala-nine,L-proline,L-serine,L-threonine,L-tryptophan andL-valine,all at 160 mg/l), the vitamins 0.036lM cyanocobalamine, 0.29lM4-aminobenzoic acid, 0.04lM biotin, 0.81lM nicotinic acid,0.21lM D-pantothenic acid (Ca salt), 0.62lM pyridoxamine di-hydrochloride, 0.29lM thiamine dichloride and 0.26lM riboflavin, and with 7.5lM FeCl2, 0.51lM ZnCl2, 0.5lM MnCl2,0.097lM boric acid, 1.46lM CoCl2, 0.015lM CuCl2, 0.1lMNiCl2, and 0.148lMNa2MoO4.

2) Bucilfus subtilis

• 相关信息 (BERGEY’S MANUAL OF Systematic Bacteriology, 2nd Edition, V3, The Firmicutes)

  Aerobes or facultative anaerobes, but a few species are described as strictly anaerobic.

• 文献4 Temporal activation of β-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool
  J Stülke，R Hanschke，M Hecker -1993:《Microbiology》

• Media and growth conditions of Bacillus subtilis
  CeIls of B. subtilis were grown at 37 "C in basal limitation medium (BLM) containing nutrients according to the type of limitation required.

• Basal limitation medium (BLM) consists of :50 mM-Tris, 15 mM- (NH4)SO4, 8 mM-MgSO4, 27 mM-KC1 and 7 mM-sodium citrate (pH 7.5).

• The following substances were added from separate stock solutions: CaCl2, to 2 mM, FeSO4, to 1 mM, MnSO4, to 10 μM and potassium glutamate to 4-5 mM.

• Amino acid starvation medium (ASM) contained 0.1 % glucose, 0.4 mM KH2PO4, and 12.8 ug/ml of each of the required amino acids according to the auxotrophies of the strains.

• Glucose limitation medium (GLM) was supplemented with 0.05 % glucose, 0.4 mM-KH2PO4, and 160 μg/ml of each of the required amino acids. All media were balanced to support growth to an OD of 1.2. The actual starvations were regularly verified by the re-addition of the exhausted nutrient and re-initiation of growth.

待续更新。。。