胞内游离氨基酸具有周转快的特点，其13 C同位素丰度能快速反映胞内代谢状态的变化。但胞内游离氨基酸的浓度很低，现有的基于气相色谱-质谱联用全扫描模式的13 C同位素丰度检测方法不能满足要求。本研究考察理论上检测精度更高的选择离子监测方法在胞内游离氨基酸13 C同位素丰度分析中应用的可能性。首先在全扫描模式下分析了不同氨基酸的断裂规律，找出与每种氨基酸对应的特征碎片，建立起包含有16种胞内游离氨基酸的特征碎片库。利用此特征碎片库，在样品分析时只需检测特定m/z处的信号，从而实现选择离子监测，提高信号质量。对标准品的检测结果表明，与全扫描模式相比，本方法的信噪比、测量精度和准确性分别提高了17倍、2倍和3.8倍。在对辅酶Q10生产菌株样品的分析中，本方法成功检测出8种胞内游离氨基酸的同位素丰度。%13 C isotopic abundance of intracellular free amino acid with a characteristic of fast- turnover can quickly reflect changes in intracellular metabolic state. But the concentration of intracellular free amino acid is low, the existed 13 C isotope detection method based on GC-MS can not satisfy the requirement with full scan mode. In this study, the selected ion monitoring method was used to detect accuracy higher likelihood of analysis of 13 C isotopic abundance of free intracellular amino acid. First, in the full scan mode we analyzed of the fracture law of different amino acids, found the feature corresponding to each amino acid fragments, and established 16 kinds of free intracellular amino acids characteristic fragment library. Then using this characteristic fragment library, only specific m/z signal was detected in sample analysis, which realized the selected ion monitoring and improved the quality of signal. The results of amino acid standards showed that the signal-to-noise ratio, measurement precision and accuracy were improved by 17, 2. 0 and 3. 8 times compared with the full scan mode. In the analysis of coenzyme Q10 producing strains of samples, this method was successfully used to detect isotopic abundance of 8 kinds of free intracellular amino acids. This method plays an important role in the detection of 13 C isotopic abundance of the intracellular free amino acid in cell metabolism research.
展开▼
