質量分析装置を用いるタンパク質中D型アミノ酸残基の同定

Abstract

加齢後の組織中タンパク質内部の様々な部位に存在すると考えられる異性化Aspを同定するためには，キラル誘導体分離法，ジアステレオマー誘導体化法など煩雑な研究手法の組み合わせが一般的であった．これに対して，著者らの研究室では質量分析装置を『質量検知装置』として用い，液体クロマトグラフィーと組み合わせた手法を開発，改良して簡便・迅速にタンパク質中の異性化Asp部位を決定することに成功した．本手法では，2段階の酵素消化に応じて生成する合計4つの試料を同条件のnano-scale液体クロマトグラフィー連結型の質量分析装置へと導入する．各ペプチド断片中でAsp異性体に準じた酵素の作用による分子量変化が生じるため，質量で描くクロマトグラムから各々対応するペプチド断片のピークが消失する．これを利用することで各Asp異性化部位の同定及びAsp異性体の区別を同時に行うことが可能となった.

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 β-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 β-Asp in protein without a set of complicated methods and instruments.