Goodlett D R, Abuaf P A, Savage P A, Kowalski K A, Mukherjee T K, Tolan J W, Corkum N, Goldstein G, Crowther J B
Immunobiology Research Institute, Annandale, NJ 08801, USA.
J Chromatogr A. 1995 Jul 21;707(2):233-44. doi: 10.1016/0021-9673(95)00352-n.
A high-performance liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method is presented that allows rapid and accurate determination of amino acid chiral purity in a peptide. Peptides are hydrolyzed in hydrochloric acid-d1/acetic acid-d4 and then converted to diastereomers by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, Marfey's reagent). Mixtures of D- and L-amino acid diastereomeric pairs are resolved in one chromatographic separation using conventional reversed-phase high-performance liquid chromatography. Hydrolysis in a deuterated solvent is necessary because the original ratio of D-/L-amino acids present in a peptide changes during acid hydrolysis due to racemization. Peptide hydrolysis in deuterated acids circumvents this problem by labeling each amino acid that racemizes with one deuterium at the alpha-carbon. An increase in molecular mass of one atomic mass unit allows racemized amino acids to be distinguished from non-racemized amino acids by mass spectrometry. This procedure was used to determine the chiral purity of each amino acid in a purified, hexapeptide by-product (Arg-Lys-Lys-Asp-Val-Tyr) present in a kilogram batch of the synthetic pentapeptide, thymopentin (Arg-Lys-Asp-Val-Tyr).
本文介绍了一种高效液相色谱 - 电喷雾电离 - 质谱（LC - ESI - MS）方法，该方法能够快速、准确地测定肽中氨基酸的手性纯度。肽在盐酸 - d1 / 乙酸 - d4中水解，然后用1 - 氟 - 2,4 - 二硝基苯基 - 5 - L - 丙氨酸酰胺（FDAA，马尔费试剂）衍生化转化为非对映异构体。使用常规反相高效液相色谱在一次色谱分离中分离D - 和L - 氨基酸非对映异构体对的混合物。在氘代溶剂中水解是必要的，因为由于外消旋作用，肽中存在的D - / L - 氨基酸的原始比例在酸水解过程中会发生变化。在氘代酸中进行肽水解通过在α - 碳上用一个氘标记每个发生外消旋的氨基酸来规避这个问题。分子量增加一个原子质量单位使得可以通过质谱法区分外消旋化的氨基酸和未外消旋化的氨基酸。该程序用于测定一批千克级合成五肽胸腺五肽（Arg - Lys - Asp - Val - Tyr）中存在的纯化六肽副产物（Arg - Lys - Lys - Asp - Val - Tyr）中每个氨基酸的手性纯度。
