Identification of D-amino Acid Residues in Proteins Using Mass Spectrometry

Homochirality of amino acid residues of protein is essential for life. For a long time, it was believed that D-amino acids were excluded from living systems, and that proteins consisted only of L-amino acids. However, recent developments in analytical techniques have led to the discovery of D-amino acids in living organisms, such as peptides and proteins. Many D-amino acids are aspartates, which are located in various sites within metabolically inactive tissues. In order to identify such aspartate residues in protein, a combination of complicated methods and instruments has been previously applied. In contrast, our laboratory has recently developed a rapid, easy and comprehensive method to identify D-Asp and beta-Asp in tissues using LC-MS/MS, thus using mass spectrometry as a "mass detection device". A total of four samples from the same tissue and proteins, generated in two steps of enzymatic digestion, are used for LC-MS/MS. The identification of each iso-Asp containing peptide peak is performed by the disappearance of the chromatogram due to the alteration of molecular weight by each enzymatic digestion. By using this methodology, it is possible to identify and distinguish each D-Asp and beta-Asp in protein without a set of complicated methods and instruments.