Kim J, Kim K
Biotechnology Research Institute, LUCKY Research Park, Dae-jeon, Korea.
Biochem Mol Biol Int. 1994 Nov;34(5):897-907.
The combination method of carboxypeptidase Y digestion and fast atom bombardment (FAB) mass spectrometry is described for the identification of C-terminal amino acid amides in peptides. Carboxypeptidase Y has amidase activity as well as exopeptidase activity in the same digestion buffer condition. Based on this concept, we develop a new technique which can definitively and easily identify the C-terminal amino acid amides. This method obviates the need for several complicated steps occurring in previous methods, but improves sensitivity, and enables exact identification of the amino acid amide by the difference of molecular mass. Analyses of carboxypeptidase Y digested peptides, not liberated free amino acid amides, were carried out by fast atom bombardment mass spectrometry. The use of truncated peptides by fast atom bombardment mass spectrometry in C-terminal amino acid amide determination gives several advantages over analyses of the liberated amino acid amides. The C-terminal amino acid amides of Allantostatin I (Leu-NH2), alpha-Melanocyte Stimulating Hormone (Val-NH2), and Ranatensin (Met-NH2) are unequivocally determined at a level of 0.90-2.3 nmol per peptide. This approach is based on entirely different principles than the previous approaches.
本文描述了一种结合羧肽酶Y消化法和快原子轰击（FAB）质谱法来鉴定肽中C端氨基酸酰胺的方法。在相同的消化缓冲液条件下，羧肽酶Y同时具有酰胺酶活性和外肽酶活性。基于这一概念，我们开发了一种新技术，能够明确且轻松地鉴定C端氨基酸酰胺。该方法避免了先前方法中出现的几个复杂步骤，提高了灵敏度，并能通过分子量差异准确鉴定氨基酸酰胺。通过快原子轰击质谱法对羧肽酶Y消化后的肽段（而非游离的氨基酸酰胺）进行分析。在C端氨基酸酰胺测定中，使用快原子轰击质谱法对截短肽段进行分析比分析游离氨基酸酰胺具有多个优势。尿囊抑素I（Leu-NH2）、α-黑素细胞刺激素（Val-NH2）和蛙皮素（Met-NH2）的C端氨基酸酰胺在每个肽段0.90 - 2.3 nmol的水平上被明确测定。该方法基于与先前方法完全不同的原理。
