JP2006322733A - タンパク質のプロテオーム定量分析方法及び装置 - Google Patents
タンパク質のプロテオーム定量分析方法及び装置 Download PDFInfo
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Abstract
【解決手段】測定対象から抽出したタンパク質混合物10を、分離操作を経ずに酵素による一斉消化や化学的切断を行ない、生成したペプチド混合物12中のマーカーペプチド14を液体クロマトグラフィー(LC)等により分離して、質量分析装置(MS)により検出する。
【選択図】図1
Description
これは、対象試料を2次元ゲル電気泳動(非特許文献1参照)により分離後、染色操作を行ない、各染色スポットの光学濃度、あるいは、予め蛍光標識したものについては、その蛍光強度を測定することで検出を行なう。それぞれの染色スポットに含まれるタンパク質の同定は、ゲル中での酵素消化により生じたペプチド断片の質量分析により行ない、得られた断片質量の組合せを、検索エンジンを用いてタンパク質データベースと照合するペプチドマスフィンガープリンティング(PMF)法(非特許文献2参照)、あるいは、質量分析計を2台直列に連結したタンデム質量分析(MS/MS)装置を用いてタンデム質量分析(MS/MS)を行ない、フラグメントパターンや部分配列情報をデータベースと比較することにより決定している(非特許文献3参照)。
これに対して、タンパク質を分離せずに消化を行ない、得られた混合物を一次元あるいは多次元の高速液体クロマトグラフィー(HPLC)により分離し、オンラインで質量分析測定を行なう(LC−MS)方法(ショットガン法)が普及されている。このような場合、一方の試料を安定同位体が豊富な培地で培養(非特許文献5参照)、あるいはICAT(Isotope Coding Affinity Tag)法(非特許文献6及び特許文献1参照)に代表される、抽出後に化学的な方法によって同位体標識された試料を、他方の試料に添加して内部標準として扱い、各消化ペプチドの相対的な変動を観測する。同時に自動MS/MS解析を行なうことで、各ペプチドからタンパク質への帰属を行ない、変動したタンパク質を決定できる。
12…ペプチド混合物
14…マーカーペプチド
16…液体クロマトグラフィー/質量分析装置(LC−MS)
Claims (6)
- 測定対象から抽出したタンパク質混合物を、分離操作を経ずに酵素による一斉消化や化学的切断を行ない、
生成したペプチド混合物中のマーカーペプチドを分離して、質量分析することを特徴とするタンパク質のプロテオーム定量分析方法。 - 一つのタンパク質に、複数のマーカーペプチドを設定することを特徴とする請求項1に記載のタンパク質のプロテオーム定量分析方法。
- 前記分離を、液体クロマトグラフィーにより行なうことを特徴とする請求項1又は2に記載のタンパク質のプロテオーム定量分析方法。
- 前記分離を、キャピラリー電気泳動により行なうことを特徴とする請求項1又は2に記載のタンパク質のプロテオーム定量分析方法。
- 前記分離を、マイクロチップにより行なうことを特徴とする請求項1又は2に記載のタンパク質のプロテオーム定量分析方法。
- 測定対象から抽出したタンパク質混合物を、分離操作を経ずに酵素による一斉消化や化学的切断を行なう切断手段と、
生成したペプチド混合物中のマーカーペプチドを分離する分離手段と、
分離されたマーカーペプチドを検出する質量分析装置と、
を備えたことを特徴とするタンパク質のプロテオーム定量分析装置。
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Cited By (2)
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JP2009080010A (ja) * | 2007-09-26 | 2009-04-16 | Hitachi High-Technologies Corp | 質量分析システム |
JP2011522210A (ja) * | 2007-06-01 | 2011-07-28 | アンセルム(アンスチチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル) | ポリペプチドを絶対定量化するための方法 |
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JP2006504971A (ja) * | 2002-11-01 | 2006-02-09 | ザ・リージェンツ・オブ・ザ・ユニバーシティ・オブ・コロラド,ア・ボディー・コーポレイト | マトリックス支援レーザー脱離イオン化−飛行時間型質量分析によるタンパク質アイソフォームの定量的解析 |
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JP2006504971A (ja) * | 2002-11-01 | 2006-02-09 | ザ・リージェンツ・オブ・ザ・ユニバーシティ・オブ・コロラド,ア・ボディー・コーポレイト | マトリックス支援レーザー脱離イオン化−飛行時間型質量分析によるタンパク質アイソフォームの定量的解析 |
Non-Patent Citations (1)
Title |
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JPN6010003185, Fagen Zhang et al., "Quantitation of human glutathione S−transferases in complex matrices by liquid chromatography/tandem", Rapid Communications in Mass Spectrometry, 2004, Volume 18 Issue 4, 491 − 498 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2011522210A (ja) * | 2007-06-01 | 2011-07-28 | アンセルム(アンスチチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル) | ポリペプチドを絶対定量化するための方法 |
JP2009080010A (ja) * | 2007-09-26 | 2009-04-16 | Hitachi High-Technologies Corp | 質量分析システム |
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