Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Chem Biodivers. 2010 Jun;7(6):1644-50. doi: 10.1002/cbdv.200900309.
The number of reports indicating the occurrence of D-amino acids in various proteins and natural peptides is increasing. For a usual detection of peptidyl D-amino acids, proteins or peptides are subjected to acid hydrolysis, and the products obtained are analyzed after cancellation of the effect of amino acid racemization during the hydrolysis. However, this method does not seem reliable enough to determine the absence or presence of a small amount of innate D-amino acids. We introduce a modification of an alternative way to distinguish true innate D-amino acids from those artificially generated during hydrolysis incubation. When model peptides (L-Ala)(3), D-Ala-(L-Ala)(2) are hydrolyzed in deuterated hydrochloric acid (DCl), only newly generated D-amino acids are deuterated at the alpha-H-atom. Both innate D-amino acids and artificially generated ones are identified by the combination of high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry equipped with a chiral column. When a peptide containing D-Phe residues was analyzed by this method, the hydrolysis-induced conversion to L-Phe was similarly identified.
越来越多的报告表明，各种蛋白质和天然肽中存在 D-氨基酸。为了通常检测肽基 D-氨基酸，将蛋白质或肽进行酸水解，并且在水解过程中消除氨基酸外消旋作用的影响之后分析获得的产物。然而，该方法似乎不足以可靠地确定少量固有 D-氨基酸的存在或不存在。我们介绍了一种替代方法的改进，以区分真正的天然内源性 D-氨基酸与在水解孵育过程中人为产生的那些。当模型肽（L-Ala）（3），D-Ala-（L-Ala）（2）在氘代盐酸（DCl）中水解时，仅新生成的 D-氨基酸在α-H-原子处被氘化。通过配备手性柱的高效液相色谱和液相色谱/串联质谱的组合来鉴定天然内源性 D-氨基酸和人为生成的 D-氨基酸。当通过该方法分析含有 D-Phe 残基的肽时，也同样鉴定了水解诱导的转化为 L-Phe。
