On-line clean-up and LC-MS analysis of primary metabolites in cell culture supernatants
On-line clean-up and LC-MS analysis of primary metabolites in cell culture supernatants
复制标题
细胞培养上清液中初级代谢物的在线净化和 LC-MS 分析
DOI：
10.1039/c7ay02034a
复制
发表时间：
2017
影响因子：
3.1
通讯作者：
Hann S
中科院分区：
文献类型：
--
作者：
Mairinger T;Tröndle J;Hanscho M;Hann S
生物技术样品的现代代谢谱分析的当前方法面临着几个挑战，例如宽的浓度范围和基质的复杂性。虽然细胞外代谢物的浓度水平的信息是非常需要的，特别是流量平衡分析，测量是由一个高度复杂的矩阵阻碍。随着时间的推移，这会影响分析方法的一般性能。在本研究中，一个自动化的清理程序，旨在通过液相色谱法结合飞行时间质谱分析的初级代谢产物的基质分离，进行了测试和优化的中国仓鼠卵巢培养物的上清液。采用均匀13C标记的酵母细胞提取物评估回收率。优化的在线净化，包括过滤和固相萃取去除基质，显示回收率为45.0%至138.7%，平均相对标准偏差为11.6%。此外，还通过精确质谱法进行了非靶向分析方法，用于基质分离的差异评估。
Current approaches to modern metabolic profiling of biotechnological samples are facing several challenges, e.g. the wide concentration ranges and the complexity of the matrix. While the information on concentration levels of extracellular metabolites is much in demand, especially for flux balance analysis, the measurement is hampered by a highly complex matrix. This affects the general performance of an analytical method over time. In the present study, an automated clean-up procedure, aiming at matrix separation for the analysis of primary metabolites via liquid chromatography coupled with time-of-flight mass spectrometry, was tested and optimized for supernatants of Chinese hamster ovary cultures. A uniformly 13C labeled yeast cell extract was employed for assessing recovery. The optimized on-line clean-up, comprising filtration and solid phase extraction for matrix removal, showed recoveries ranging from 45.0% to 138.7%, with an average of 11.6% relative standard deviation. Additionally, a non-targeted analytical approach by accurate mass spectrometry for the differential assessment of the matrix separation was performed.