黄色短杆菌中L-异亮氨酸同位素丰度及分布的分析方法研究  

Analysis of Isotope Abundance and Distribution for L-Isoleucine in Brebvibacterium flavum

作　　者：赵雅梦 范若宁 雷雯 ZHAO Ya-meng;FAN Ruo-ning;LEI Wen(Shanghai Research Institution of Chemical Industry Co.Ltd.,Shanghai 200062,China;Shanghai Professional Technology Service Platform on Detection and Application Development for Stable Isotope,Shanghai 200062,China)

机构地区：[1]上海化工研究院有限公司,上海200062 [2]上海市稳定同位素检测及应用研发专业技术服务平台,上海200062

出　　处：《分析测试学报》2024年第3期496-500,共5页Journal of Instrumental Analysis

基　　金：国家重点研发计划项目典型同位素试剂研发及科研试剂技术标准研究(2021YFF0701900);上海市2021年度“科技创新行动计划”临床质谱检测用系列同位素标记试剂的制备及应用研究(21142201600);上海市2022年度“科技创新行动计划”长三角科技创新共同体领域项目基于自主可控原材料的临床质谱试剂盒全链条开发及产业化(22002400400)。

摘　　要：随着代谢组学、蛋白质组学等生命科学领域的迅猛发展,稳定同位素标记试剂,尤其是标记氨基酸,因无放射性、与非标记化合物理化性质一致等优势得到广泛应用。该文建立了一种稳健、快速的氨基酸同位素丰度分析方法。方法采用Hypersil Gold Vanquish(100 mm×2.1 mm,1.9µm)色谱柱,以水和含0.1%甲酸的甲醇为流动相,正离子模式下进行液相色谱-高分辨质谱联用(LC-HRMS)分析;测得细菌发酵液中L异亮氨酸-15N的同位素丰度为98.58%,相对标准偏差为0.03%,可应用于不同稳定同位素(15N或^(13)C)示踪的黄色短杆菌中L-异亮氨酸同位素丰度及分布的准确测定。该方法具有简便、灵敏、稳健等优点,有望在合成生物学、同位素示踪代谢流等研究中发挥重要作用。In the rapidly advancing life science fields such as metabolomics and proteomics,stable isotope labeling reagents that are non-radioactive and have similar physiochemical properties with un⁃labeled compounds have been widely utilized.Biological fermentation is one of the major synthesis ap⁃proaches for labeled amino acids.In this study,we have established an accurate,robust,and rapid method to determine the isotope abundance of the amino acids in the fermentation broth to aid in early assessment of batch quality and optimization of fermentation conditions and amino acid yield.A Hy⁃persil Gold Vanquish column(100 mm×2.1 mm,1.9µm)with water and methanol containing 0.1%formic acid as mobile phase and a liquid chromatography-high resolution mass spectrometry(LC-HRMS)system in positive ion mode were used for the study.The isotopic abundance of L-iso⁃leucine-15N samples was determined to be 98.58%,closely matching the indicated value(>98%),with a relative standard deviation of 0.03%,demonstrating excellent accuracy and precision for the method.Then the method was successfully applied to determine the isotopic abundance and distribu⁃tion of L-isoleucine in Brevibacterium flavum labeled with 15N or ^(13)C.The proposed method is simple to perform,convenient,highly sensitive,and robust,holding wide application potentials in syn⁃thetic biology and research in stable isotope traced metabolic pathways.

关 键 词：同位素标记氨基酸 液相色谱-高分辨质谱(LC-HRMS) 黄色短杆菌 同位素分布及丰度 

分 类 号：O657.72[理学—分析化学] O629.7[理学—化学]