13CMFA过程中GC-MS分析菌体蛋白氨基酸的13C标记丰度

基于13C标记的代谢通量分析(13CMFA)由于具有准确性和应用性已成为国际上代谢工程研究的热点，其中一个至关重要的问题就是分析菌体蛋白氨基酸的13C标记丰度信息。用含20%全标记葡萄糖（［U-13C］）和80%天然葡萄糖的合成培养基喂养维生素B12生产菌Pseudomonas denitrifican，然后在稳态条件下取20mg带13C标记菌体用1ml 6mol/L盐酸95℃水解24h得到带13C标记的菌体蛋白氨基酸；氨基酸经分离、浓缩、真空干燥、MBDSTFA衍生化后得到的TBDMS衍生物可以用气相色谱-质谱(GC-MS)进行分析，最后分析质谱图得到15种菌体蛋白氨基酸的13C标记丰度信息。成功获得菌体蛋白氨基酸13C标记丰度信息的实验方法和样品处理技术，为13CMFA在国内进一步发展提供了参考意义。

Analysis of Mass Isotopomer Distributions of Proteingenic Amino Acids Used GC-MS during 13CMFA

Abstract ¹³C metabolic flux analysis (13CMFA) have been the research hotspots of metabolic engineering internationally due to its accuracy and applicability.It is vital that the measurement of 13C labelling pattern of proteinogenic amino acids for ¹³C metabolic flux analysis.To acquire ¹³C-labelling proteinogenic amino acids,Pseudomonas denitrifican which products Vitamin B12 was firstly fed with mimimal culture medium contained 20% U-¹³C and 80% natural glucose，after the culture reached a steady state, then about 20 mg biomass was hydrolyzed by 1 ml of 6 mol/L hydrochloric acid for 24h at 110℃.Then amino acids was separated,concentrated, evaporated in a vacuum, and derivatized with MBDSTFA, TBDMS-derivatized amino acids can be observed by GC-MS last we get ¹³C labelling pattern of fifteen aminio acids through mass spectrum.The experimental methods and sample preparation offers referential value for the development of 13C metabolic flux analysis in our courtry.