衍生Aldononitrile乙酸氨基葡萄糖和胞壁酸的微生物在土壤中残留量的测定气相色谱的检测
Summary
我们描述了一个协议aldonitrile从土壤中提取的葡萄糖和胞壁酸醋酸衍生物GC为基础的分析方法。对于澄清的化学机制，我们也提出一项战略，以确认衍生工具及电子电离后形成的碎片离子的结构。
Abstract
表征微生物的定量方法是为更广泛地了解微生物生态系统内的地位和功能的关键。微生物分析当前的策略包括传统的实验室培养相关的技术和基于直接提取和测定某些生物标志物1，2。居住在土壤中的微生物物种多样性之间很少可以培养的，所以依赖于文化的方法引进重大偏差，缺席的生物标志物分析的限制。
葡萄糖，半乳糖，甘露和胞壁酸已担任活着和死去的微生物质量的措施，这些葡萄糖最丰富的胞壁酸（唯一从细菌细胞）是最重要的成分在土壤系统3 4。然而，分析知识缺乏制约的科学同行之间的广泛普及。其中所有EXI蜇分析方法，到aldononitrile乙酸酯衍生GC为基础的分析，作为不错的选择，以最佳的平衡精度方面出现的，灵敏度，简单，良好的色谱分离，样品储存5时的稳定性。
在这里，我们提出了一个详细的协议为葡萄糖和胞壁酸的可靠和相对简单的分析，从土壤后，他们转换到aldononitrile醋酸。该协议主要包括四个步骤：酸消化，样品净化，衍生化和气相色谱法测定。根据修改前的出版物6，7分步过程。此外，我们提出了一项战略，以结构验证的分子离子和电子电离后形成的衍生离子碎片。我们采用的GC-EI-MS-SIM卡，的LC-ESI-TOF-MS的同位素标记试剂来确定分子量aldononitrile醋酸衍生物氨基葡萄糖和MUR聚酰胺酸;我们用同位素标记的衍生工具的大规模转移，在调查每个衍生物8离子碎片离子谱。在另外的理论澄清，验证衍生的分子离子和离子碎片将是有益的研究人员使用的δ13 C或离子碎片，这些生物标志物在生物地球化学研究，9，10。
Protocol
Discussion
提出的GC基于衍生aldononitrile乙酸氨基葡萄糖和胞壁酸的分析方法，提供了一个相对快速的方法来量化这些氨基酸糖，从土壤中提取。衍生品是化学性质稳定，可以在一次分析确定。该方法不仅限于土壤样品，并可以简化从非土壤基质的样品。
在这种方法中使用的真空泵是建立耐酸。我们进一步建议设立一个基本的陷阱，以保护泵，蒸发6M HCl溶液时。必须小心在衍生步骤，严?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由能源部大湖生物能源研究中心（DE-FC 02-07ER64494科学能源部的BER办公室）赠款支持。张旭东博士和他的小组成员有用的技术讨论和宝贵意见，最后敲定协议，我们对此表示感谢。
Materials
| Name of the reagent | Company | Catalogue number |
| Muramic acid | Sigma-Aldrich | M2503-25MG |
| D-(+)-glucosamine hydrochloride | Sigma-Aldrich | G1514-100G |
| N-methyl-D-glucamine | Sigma-Aldrich | M2004-500G |
| Myo-inositol | Fisher Scientific | A307003G025 |
| Methanol (dry) | Acros organics | AC326950010 |
| 4-dimethylamino-pyridine | Acros organics | AC148270050 |
| Ethyl acetate | VWR International | BJGC100-4 |
| Hydroxlamine hydrochloride | Fisher Scientific | H330-100 |
| Pyridine | Fisher Scientific | P368-500 |
| Acetic anhydride | Fisher Scientific | A10-100 |
| Dichloromethane (Methylene chloride) | Fisher Scientific | D37-500 |
| Hexane | Fisher Scientific | H303-4 |
| Hydrochloric acid (6M) | Fisher Scientific | S456-4 |
| Hydroxylamine hydrochloride-15N | Icon services | IN5280 |
| Acetic anhydride-2H (D6C4O3) | Acros organics | AC174670050 |
| D-glucose-U-13C | Cambridge isotope lab | CLM-1396-1 |
| Ammonium sufate-15N | Cambridge isotope lab | NLM-713-1 |
| Name of the equipment | Company | Type |
| Rapid-Vap | LabConco | 790002 |
| Vacum pump | KNF Neuberger | D-79112 |
| Hydrolysis flask | Fisher Scientific | 06 423A |
| Derivatization microvial | Fisher Scientific | 06-100E |
| GC | Hewlett-Packard | 6890 |
| MS | Hewlett-Packard | 5972 |
| LC-ESI-TOF-MS | Agilent | An Agilent 1200 series HPLC system coupled to an Agilent LC/MSD-TOF |
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