Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany.
Institute of Biotechnology, RWTH Aachen University, 52062, Aachen, Germany.
Anal Bioanal Chem. 2021 May;413(12):3253-3268. doi: 10.1007/s00216-021-03261-3. Epub 2021 Mar 31.
With the utilization of small-scale and highly parallelized cultivation platforms embedded in laboratory robotics, microbial phenotyping and bioprocess development have been substantially accelerated, thus generating a bottleneck in bioanalytical bioprocess sample analytics. While microscale cultivation platforms allow the monitoring of typical process parameters, only limited information about product and by-product formation is provided without comprehensive analytics. The use of liquid chromatography mass spectrometry can provide such a comprehensive and quantitative insight, but is often limited by analysis runtime and throughput. In this study, we developed and evaluated six methods for amino acid quantification based on two strong cation exchanger columns and a dilute and shoot approach in hyphenation with either a triple-quadrupole or a quadrupole time-of-flight mass spectrometer. Isotope dilution mass spectrometry with CN labeled amino acids was used to correct for matrix effects. The versatility of the methods for metabolite profiling studies of microbial cultivation supernatants is confirmed by a detailed method validation study. The methods using chromatography columns showed a linear range of approx. 4 orders of magnitude, sufficient response factors, and low quantification limits (7-443 nM) for single analytes. Overall, relative standard deviation was comparable for all analytes, with < 8% and < 11% for unbuffered and buffered media, respectively. The dilute and shoot methods with an analysis time of 1 min provided similar performance but showed a factor of up to 35 times higher throughput. The performance and applicability of the dilute and shoot method are demonstrated using a library of Corynebacterium glutamicum strains producing L-histidine, obtained from random mutagenesis, which were cultivated in a microscale cultivation platform.
利用嵌入在实验室机器人中的小规模和高度并行化的培养平台，微生物表型和生物工艺开发得到了极大的加速，从而在生物分析生物工艺样品分析中产生了瓶颈。虽然微尺度培养平台允许监测典型的过程参数，但只有有限的关于产品和副产物形成的信息，而没有全面的分析。液相色谱-质谱联用可以提供这样全面和定量的见解，但通常受到分析运行时间和吞吐量的限制。在这项研究中，我们开发并评估了六种基于两个强阳离子交换柱和稀释-进样方法的氨基酸定量方法，与三重四极杆或四极杆飞行时间质谱仪联用。用 CN 标记的氨基酸进行同位素稀释质谱法可用于校正基质效应。通过详细的方法验证研究，证实了该方法在微生物培养上清液代谢物分析中的多功能性。使用色谱柱的方法显示出约 4 个数量级的线性范围、足够的响应因子和低定量限（7-443 nM），适用于单个分析物。总的来说，所有分析物的相对标准偏差都相当，未缓冲和缓冲介质的相对标准偏差分别为<8%和<11%。分析时间为 1 分钟的稀释-进样方法具有类似的性能，但具有高达 35 倍的更高吞吐量。使用从随机诱变获得的生产 L-组氨酸的 Corynebacterium glutamicum 菌株文库，在微尺度培养平台中进行培养，证明了稀释-进样方法的性能和适用性。
