Gathungu Rose M, Oldham John T, Bird Susan S, Lee-Parsons Carolyn W T, Vouros Paul, Kautz Roger
Barnett Institute of Chemical and Biological Analysis and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA; ; Tel: +1-617-373-8211.
Anal Methods. 2012 Jan 1;4(5):1315-1325. doi: 10.1039/C2AY05803K. Epub 2012 Apr 12.
Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for obtaining natural products of medical relevance. Cultures can be optimized for high yields of desired metabolites using rapid profiling assays such as HPLC. We describe an approach to establishing a rapid assay for profiling cell culture expression systems using a novel microscale LC-UV-MS-NMR platform, designed to acquire both MS and NMR each at their optimal sensitivity, by using nanosplitter MS from 4 mm analytical HPLC columns, and offline microdroplet NMR. The approach is demonstrated in the analysis of elicited Eschscholzia californica cell cultures induced with purified yeast extract to produce benzophenanthridine alkaloids. Preliminary HPLC-UV provides an overview of the changes in the production of alkaloids with time after elicitation. At the time point corresponding to the production of the most alkaloids, the integrated LC-MS-microcoil NMR platform is used for structural identification of extracted alkaloids. Eight benzophenanthridine alkaloids were identified at the sub-microgram level. This paper demonstrates the utility of the nanosplitter LC-MS/microdroplet NMR platform when establishing cell culture expression systems.
植物细胞和组织培养是一种可扩展且可控的替代完整植物的方法，用于获取具有医学相关性的天然产物。可以使用高效液相色谱（HPLC）等快速分析方法对培养物进行优化，以实现所需代谢物的高产率。我们描述了一种使用新型微尺度液相色谱 - 紫外 - 质谱 - 核磁共振（LC - UV - MS - NMR）平台建立细胞培养表达系统快速分析方法的途径，该平台旨在通过使用来自4毫米分析型HPLC柱的纳米分流器质谱和离线微滴核磁共振，分别以其最佳灵敏度获取质谱和核磁共振数据。该方法在分析用纯化酵母提取物诱导产生苯并菲啶生物碱的松叶烟细胞培养物中得到了验证。初步的HPLC - UV提供了诱导后生物碱产量随时间变化的概况。在对应于生物碱产量最高的时间点，集成的LC - MS - 微线圈核磁共振平台用于对提取的生物碱进行结构鉴定。在亚微克水平鉴定出了八种苯并菲啶生物碱。本文展示了纳米分流器LC - MS/微滴核磁共振平台在建立细胞培养表达系统时的实用性。
