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ナノスケール液体クロマトグラフィー/イオンモビリティスペクトロメトリー/タンデム質量分析によるリン酸化ペプチド大規模解析に関する研究

小形, 公亮 京都大学 DOI:10.14989/doctor.r13485

2022.03.23

概要

タンパク質の可逆的リン酸化修飾は細胞のシグナル伝達を担う主要な翻訳後修飾の一つである。ナノ液体クロマトグラフィー/タンデム質量分析(nanoLC/MS/MS)を用いたリン酸化プロテオミクスの発展により、リン酸化修飾部位やその増減を大規模に明らかにすることが出来るようになった。試料より抽出された全タンパク質をペプチド断片に酵素消化して一斉解析するリン酸化プロテオミクスでは数万のリン酸化修飾部位を一挙に解析できるが、一方で、広い濃度ダイナミックレンジを持ち、かつ親水性から疎水性にわたる幅広い物性を有するペプチド群を余すところなく回収し、完全に分離・検出することは困難を極める。またリン酸化ペプチドには同一のペプチド配列の異なる部位にリン酸化修飾を持つ“位置異性体”が存在するが、これら異性体は全く同一の質量をもつため質量分析では区別がつかないうえ、化学的物性が近しいため分離することも容易ではない。したがって、生体試料から得られるリン酸化ペプチドの分離分析における溶出挙動メカニズムの解明、さらには抽出法・分離法の高性能化が、リン酸化プロテオーム解析のためには必須である。

本研究ではまず、リン酸化ペプチドの逆相クロマトグラフィーにおける保持機構の解明を目的とし、様々な特性を有する逆相シリカカラムを用い、複数の溶液条件におけるリン酸化ペプチドの保持挙動を調べた。その結果、リン酸化ペプチドの保持挙動は、リン酸基の付与によるペプチドのnet chargeの減少と、linear solvent strength theory(LSStheory)で説明可能であった。

プロテオミクスでは試料の精製のため逆相固相抽出カラムが用いられる。しかし、標準的な抽出条件では親水性ペプチドを回収できず失ってしまう。一方で親水性ペプチドを回収するために分離モードを変更すると疎水性ペプチドの回収率が損なわれる。そこで逆相カラムへの試料添加時に、カラムを4℃まで冷却し、かつ有機溶媒を使用しないことでペプチドの固定相への保持を増強し、標準プロトコルと比較して2.9倍の親水性ペプチドと6.1倍の親水性リン酸化ペプチドを同定することに成功した。この際、疎水性ペプチドの回収率の低下は見られず、さらに定量再現性が向上することが確認できた。

続いて、逆相固相抽出カラムを利用したナノスケールの同重体(tandemmasstag,TMT)固相標識法を開発し、高感度な多重定量リン酸化プロテオミクスのワークフローを確立した。数十から数百ナノグラムのリン酸化ペプチドに対して最適化されたプロトコルを用いることで、従来の溶液反応を用いるプロトコルと比較して、約10倍の感度向上が得られた。本標識法を用い、50µgのHeLaタンパク質から分子標的薬セルメチニブで制御されるリン酸化プロテオームの定量に成功した。さらに、invitroキナーゼ反応を利用して標的となるリン酸化ペプチドを人工的に生成し,これを同位体標識チャンネルの1つにスパイクして検出性を高めるモチーフ標的リン酸化プロテオミクスに本法を応用し、数十µgの出発物質から数千以上のチロシンリン酸化部位を定量することに成功した。近年、クロマトグラフィーにおける保持時間、質量分析におけるm/zに加えてイオンモビリティスペクトロメトリーにおける分離がプロテオミクスのための新たな分離場として注目されてきている。NanoLCとlinear solvent strength theory(LSS theory)による二次元分離を検討した結果、LCとTIMSを組み合わせることで一時間あたり最大3300のピークキャパシティを得ることが可能であった。

加えて、LC/TIMS/Q/TOFシステムにより、総測定時間を増加させることなく、プロテオーム解析における同重体標識に基づく定量精度を大幅に向上させることができることを示した。また、LCとMSの間にTIMSを挿入すると,LCとMSを補完するペプチド構造情報が得られる。しかし,TIMSの有用性を最大限に引き出すためには,リン酸化ペプチドの衝突断面積(CCS)を正確に予測し,リン酸基がペプチドのCCS値にどのような影響を与えるかを知る必要がある。そこで、TIMSを用いて,モノリン酸化ペプチドとそれに対応する非リン酸化ペプチドの4,433組のイオンのCCS値を系統的に解析した。結果、リン酸化によって質量が80Da増加するにもかかわらず、モノリン酸化ペプチドイオンの約3分の1は、リン酸化されていないイオンよりも小さなCCS値を示すことを明らかにした。この減少は塩基性官能基をより多く持つペプチドで顕著であったことから、リン酸基と塩基性官能基の分子内相互作用の形成がCCSの減少に関係していることを反映していると考えられた。

以上、リン酸化ペプチドの分離分析における分離メカニズムの解明に向け、逆相クロマトグラフィーとTIMSにおけるリン酸化ペプチドの分離挙動を説明するモデルを確立した。さらに、逆相固相抽出法を用いた試料前処理法の開発により、より広範な物性のリン酸化ペプチドの、より高い感度での解析を達成した。本研究により、リン酸化プロテオミクスの高性能化、さらなる適用拡大が期待される。

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