A nonempirical method using LC/MS for determination of the absolute configuration of constituent amino acids in a peptide: Combination of Marfey's method with mass spectrometry and its practical application
A nonempirical method using LC/MS for determination of the absolute configuration of constituent amino acids in a peptide: Combination of Marfey's method with mass spectrometry and its practical application
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DOI：
10.1021/ac970289b
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发表时间：
1997-12-15
影响因子：
7.4
通讯作者：
Harada, K
中科院分区：
文献类型：
--
作者：
Fujii, K;Ikai, Y;Harada, K
我们之前的论文中提出的“改进的马尔菲法”已发展为使用液相色谱/质谱（LC/MS）非经验性地确定肽中组成氨基酸的绝对构型。为了建立该方法，我们必须解决以下三个问题：（1）阐明被选作色谱技术的马尔菲法的局限性及其分离机制，因为该提出的方法依赖于用1 - 氟 - 2,4 - 二硝基苯基 - 5 - L - 丙氨酰胺（L - FDAA）衍生化的氨基酸的洗脱顺序来确定其绝对构型；（2）如何有效地将马尔菲法与质谱结合，以便在没有标准样品的情况下检测和鉴定所需的氨基酸；（3）如何从肽样品中的L - 或D - 氨基酸获得相应的对映体。在之前的一篇论文中，我们研究了问题1，并最终根据提出的分离机制描述了马尔菲法用于阐明所需氨基酸洗脱顺序的合理应用指南。在本文中，我们进一步研究了剩下的两个问题。由于原始衍生试剂L - FDAA衍生的氨基酸由于可能的热不稳定性和低疏水性，对于使用任何接口的LC/MS分析灵敏度较差，我们选择电喷雾电离和多孔玻璃快速原子轰击（Frit - FAB）作为接口，并开发了1 - 氟 - 2,4 - 二硝基苯基 - 5 - L - 亮氨酰胺（L - FDLA）来替代L - FDAA作为一种新的衍生试剂，以便将马尔菲法与质谱结合。此外，我们引入了一种使用1 - 氟 - 2,4 - 二硝基苯基 - 5 - DL - 亮氨酰胺（DL - FDLA）的外消旋化程序，即“DL - FDLA衍生化”，来替代从L - 或D - 氨基酸获得相应对映体的常规化学外消旋化方法。因此，我们建立了一种使用LC/MS的非经验性方法，即“改进的马尔菲法”。该方法已成功应用于蓝藻产生的微囊藻毒素LR中组成氨基酸的表征。
The ''advanced Marfey's method'' proposed in our preceding paper has been developed to nonempirically determine the absolute configuration of constituent amino acids in a peptide using liquid chromatography/mass spectrometry (LC/MS). For the establishment of this method, we had to resolve the following three problems: (1) elucidation of the limitation of Marfey's method, which is chosen as the chromatography technique, and its separation mechanism, because this proposed method relies on the elution order of an amino acid derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-FDAA) to determine its absolute configuration; (2) how to effectively combine Marfey's method with mass spectrometry in order to detect and identify a desired amino acid without a standard sample; and (3) how to obtain the corresponding enantiomer from either the L- or D-amino acid in a peptide sample. In a preceding paper, we investigated problem 1 and finally described the rational application guideline for Marfey's method to elucidate the elution order of a desired amino acid according to the proposed separation mechanism. In this paper, we further investigated the two remaining problems. Because the sensitivity of the amino acids derivatized with the original derivatizing reagent, L-FDAA was poor for LC/MS analysis using any interfaces due to their possible thermal instability and low hydrophobicity, we chose electrospray ionization and frit-fast atom bombardment (Frit-FAB) as the interface and developed 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) instead of L-FDAA as a new derivatizing reagent in order to combine Marfey's method with mass spectrometry. Furthermore, we introduced a racemization procedure using 1-fluoro-2,4-dinitrophenyl-5-DL-leucinamide (DL-FDLA), the ''DL-FDLA derivatization'', instead of the conventional chemical racemization for obtaining the corresponding enantiomer from either the L-or D-amino acid. Thus, we have established a nonempirical method using LC/MS, the ''advanced Marfey's method''. The method was successfully applied to the characterization of constituent amino acids in microcrystin LR produced by cyanobacteria.