Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
Methods Mol Biol. 2024;2821:71-82. doi: 10.1007/978-1-0716-3914-6_6.
Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.
氨基酸分析是一种准确的方法，用于组成和定量分析多肽和这些合成肽。与质谱结合使用，它可以在缀合和免疫之前可靠地控制肽的质量和数量。最初，肽在气相中用 6-M HCl 在 110°C 下水解 20-24 小时，然后通过色谱法分析所得氨基酸，其中最可靠的形式是离子交换色谱法，带有柱后茚三酮衍生化。根据水解条件的不同，色氨酸会被破坏，半胱氨酸也是如此，除非进行衍生化，酰胺、谷氨酰胺和天冬酰胺分别脱酰胺为谷氨酸和天冬氨酸。建议使用三种不同的计算结果方法，并且考虑到上述限制，通常可以获得优于 5%的定量结果。
