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シングルセルメタボロミクスに資する液体クロマトグラフィー質量分析基盤技術の開発

中谷, 航太 NAKATANI, Kohta ナカタニ, コウタ 九州大学

2020.03.23

概要

近年,次世代シーケンサー技術の急速な発展によってシングルセルレベルでの解析が可能になり,細胞周期,細胞老化,確率論的分布などの影響を受け,生命システムには多種多様な個性を持つ細胞がヘテロに存在することが分かってきた.そして,特定の細胞が持つ個性が生命現象に重要な役割を果たしていることが明らかになりつつある.生命システムに内在するヘテロな細胞集団をシングルセルの解像度で解析することにより,アンチエイジングやがん治療などの医療戦略に有意義な洞察が得られることが期待されている.また,近年,分子階層を跨いだ生命システムの制御機構が報告されてきたことで,各階層オミクスの重要性が高まりつつある.生命システムのより良い理解のためには各階層の情報を 1細胞レベルで取得する技術の開発が急務である.しかしながら,シングルセルメタボロミクスの解析法は,他のシングルセルオミクス解析と比べて測定技術は未だ発展途上の段階である.典型的な動物細胞のような小さな細胞の 1 細胞メタボロミクスでは,超微量試料の分析が要求されるため,感度が課題となる.また,1 細胞試料は 1 度しか分析することができないため,単一分析における網羅性も課題となる.そこで本研究では,これらの課題を解決する液体クロマトグラフィー質量分析 (LC/MS) の基盤技術を開発することを目的とした.

第二章では,感度不足という問題を解決するために,カラム内径の小さな nano-LC カラムを自作し,これを用いた分離系のダウンサイジングによる高感度 nano-LC/MS 分析系の開発を行った.さらに,開発した手法を HeLa 細胞のシングルセルメタボロミクスに適用し,高感度 nano-LC/MS システムが典型的な動物細胞のシングルセルメタボロミクスに利用できることを実証した.すなわち,感度という課題に対して,分離系のダウンサイジングによる高感度 nano-LC/MS 法という解決策を提示した.

第三章では,網羅性の課題を解決するために,単一分析による網羅的な親水性メタボローム分析手法の開発に取り組んだ.利用可能な LC カラムのスクリーニングにより,アミノ基混合ポリマーカラムの可能性を見出し,これを用いて詳細に LC 条件の最適化を行った.その結果,各 LC 条件が有機的に作用し,親水性相互作用クロマトグラフィー (HILIC) と陰イオン交換クロマトグラフィー (AEX) が連続段階的に作用する新しいクロマトグラフィー分離手法を見出すことに成功した. 当該手法を unified HILIC/AEX 法と命名し,その分析性能を従来法と比較した.その結果,本手法は単一分析において最も網羅性の高い親水性メタボローム分析法であることが実証された.すなわち,シングルセルメタボロミクスが抱える網羅性という課題に対して,unified HILIC/AEX/MS 法という解決策を提案した.

本博士論文では,シングルセルメタボロミクスが抱える感度と網羅性という 2 つの課題に対して,それぞれ個別の解決策を提案した.今後,提案した 2 つの解決策を統合,すなわち,高感度 nano-unified HILIC/AEX/MS 法を構築することで高深度のシングルセルメタボローム情報の取得が実現可能になることが期待される.

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