リボソームタンパク質をバイオマーカーとしたマトリックス支援レーザー脱離イオン化質量分析法によるバクテリアの迅速同定及び分類法の開発

摘要

The rapid identification of bacteria has become of importance in various fields, such as food industries, biomedical assays, and environmental analyses. In this study, a simple, rapid, and highly reliable method for the identification and classification of bacteria by matrix-assisted laser desorption/ioniza-tion-mass spectrometry (MALDI-MS) was developed. In this method, ca. 50 different kinds of ribosomal proteins were used as biomarkers for bacteria characterization. To verify the actual state of sequence information of ribosomal proteins registered in the protein databases, a simple intact protein analysis using only cell lysate without the purification of ribosomal proteins was developed. Through the characterization of ribosomal proteins of genome-sequenced lactic acid bacteria (LAB), a guideline for selecting reliable biomarkers used for the rapid identification of LAB by MALDI-MS was proposed. Although the amino acid sequences of ribosomal proteins is highly conserved, slight sequence variations of some ribosomal proteins can occur at the strain level, reflecting different phylogenetic evolution rates. Such sequence variations could be detected as mass differences in the MALDI mass spectra. The profile of the mass differences of ribosomal proteins was processed by cluster analysis, and generating a phylogenetic tree. Using this method, the phylogenetic classification of a total of 16 strains of Pseudomonas putida was demonstrated. The classification result of P. putida strains was highly comparable to those based on a conventional DNA sequence analysis.%バクテリアを迅速簡便かつ正確に同定あるいは分類するrn分析手法の開発は，食品，医療．及び環境など様々な分野rnで強く求められている．これまで，遺伝子型の違いに基づrnく信頼性の高いバクテリア分析には，DNA塩基配列を解rn析する方法が用いられ，迅速性が要求される場合には，生rn化学的な簡易検査や質量分析法を利用した化学分類法などrnの適用が検討されてきた．しかし，迅速簡便性と正確さをrn兼ね備えたバクテリアの分析手法は確立されておらず，特rnにバクテリアの機能や毒性を特性化する上で重要となる，rn最も詳細な分類区分である「株レベル」での分類は，いずrnれの手法でも容易ではなかった．