JP5735500B2 - エネルギー代謝の極性代謝物を分析するための方法 - Google Patents
エネルギー代謝の極性代謝物を分析するための方法 Download PDFInfo
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Description
i)生物学的試料に含まれる細胞の即時破壊を可能にする条件下で相分離剤と揮発性中性アンモニウム塩とを含む抽出緩衝液を用いて該生物学的試料を抽出すること、
ii)工程i)で得た抽出物に含まれる極性代謝物をクロマトグラフィーにより分離すること、および
iii)工程ii)で得た、分離した極性代謝物を分析すること
を含む上記方法に関する。
a)水相のスピン遠心分離(spin centrifugation)、
b)工程a)で得た上清の凍結乾燥、および
c)工程b)で得た凍結乾燥物の、イオン対形成剤(ion pair generating agent)を含む分析緩衝液への溶解
により処理する。
a)プロパンジオールまたはエタンジオールを含む予冷したクエンチング溶液からなる下相、不活性相分離剤からなる疎水性中間相、および生物学的試料からなる上相を含む遠心分離バイアルの提供、ただし該下相は該中間相よりも密度が高く、かつ該中間相は該上相よりも密度が高いものとする、ならびに
b)生物学的試料に含まれる細胞性物質を前記上相から前記下相へ移動させるための、前記バイアルの遠心分離
を含む上記方法にも関する。
[1] 極性代謝物を分析するための方法であって、以下:
i)生物学的試料に含まれる細胞の即時破壊を可能にする条件下で、相分離剤と揮発性中性アンモニウム塩とを含む抽出緩衝液を用いて該生物学的試料を抽出すること、
ii)工程i)で得た抽出物に含まれる極性代謝物をクロマトグラフィーにより分離すること、および
iii)工程ii)で得た、分離した極性代謝物を分析すること
を含む上記方法。
[2] 前記相分離剤がジクロロメタンを含む、上記[1]記載の方法。
[3] 前記揮発性アンモニウム塩が酢酸アンモニウム、ギ酸アンモニウム、または炭酸水素アンモニウムである、上記[1]または[2]記載の方法。
[4] 前記揮発性中性アンモニウム塩の濃度が少なくとも0.5 Mである、上記[1]〜[3]のいずれかに記載の方法。
[5] 前記の工程i)における生物学的試料の抽出を抽出温度-20〜-80℃で実施する、上記[1]〜[4]のいずれかに記載の方法。
[6] 前記抽出物を、工程ii)に先立って以下の工程:
a)水相のスピン遠心分離、
b)工程a)で得た上清の凍結乾燥、および
c)工程b)で得た前記凍結乾燥物の、イオン対形成剤を含む分析緩衝液への溶解
により処理する、上記[1]〜[5]のいずれかに記載の方法。
[7] 前記イオン対形成剤が、トリブチルアンモニウム陽イオン、トリエチルアンモニウム陽イオン、トリプロピル-アンモニウム陽イオンまたはn-ヘキシルアンモニウム陽イオンである、上記[1]〜[6]のいずれかに記載の方法。
[8] 前記クロマトグラフィーがUPLCまたはHPLCである、上記[1]〜[7]のいずれかに記載の方法。
[9] 前記の工程iii)で得た分離した極性代謝物の分析が質量分析を含む、上記[1]〜[8]のいずれかに記載の方法。
[10] 前記質量分析の方法がESI-MS/MSである、上記[9]記載の方法。
[11] 前記生物学的試料が細胞懸濁液である、上記[1]〜[10]のいずれかに記載の方法。
[12] 細胞性物質を含む生物学的試料をクエンチするための方法であって、以下の工程:
a)プロパンジオールまたはエタンジオールを含むクエンチング溶液からなる下相、不活性相分離剤からなる中間相、および生物学的試料からなる上相を含む遠心分離バイアルを提供する工程、ただし該下相は該中間相よりも高い密度を有し、かつ該中間相は該上相よりも高い密度を有する、ならびに
b)前記生物学的試料に含まれる細胞性物質を前記上相から前記下相へ移動させるために、前記バイアルを遠心分離する工程
を含む上記方法。
[13] 前記下相が-20〜-80℃の温度を有する、上記[12]記載の方法。
[14] 上清を遠心分離後に前記下相から除去し、かつ同位体で標識した標準物質をこの下相に加える、上記[12]または[13]記載の方法。
[15] 前記細胞懸濁液を、工程i)に先立って上記[12]〜[14]のいずれかに記載した通りにクエンチングに供する、上記[11]記載の方法。
[16] 前記生物学的試料が膜上で増殖させた接着細胞の培養物である、上記[1]〜[10]のいずれかに記載の方法。
[17] 前記の膜上で増殖させた接着細胞の培養物を、工程i)に先立って、-20〜-80℃の温度を有する上記[1]〜[4]のいずれかに記載した抽出緩衝液に該膜を即時移動させることによりクエンチングに供する、上記[16]記載の方法。
[18] 前記の膜上で増殖させた接着細胞の培養物を、工程i)に先立って、液体窒素中に該膜を即時移動させることによりクエンチングに供する、上記[16]記載の方法。
[19] 前記生物学的試料が組織試料である、上記[1]〜[10]のいずれかに記載の方法。
[20] 前記組織を、工程i)に先立って、該組織を-20〜-80℃の温度まで即時冷凍することにより、または液体窒素を適用することによりクエンチングに供する、上記[18]記載の方法。
上述した全ての参考文献は、その全開示内容ならびに上記説明箇所において明確に言及したその具体的な開示内容に関して参照により本明細書に含まれるものとする。
a)細胞懸濁液
350μLのプロパン1,2-ジオールを、5個のスチールビーズを予め充填した多数の抽出バイアルに加え、-80℃まで冷却した。その後、350μLのシリコン油を各バイアルに加えてから、次回使用時まで-80℃で保存した。1ミリリットルという典型的な容量の水性細胞懸濁液を油層の上に注意深く加え、この試料を即時15000×gで2分間、0℃にて遠心分離した。続いて、該試料を即時ドライアイス上に置き、上相を除去した。その後、1.5 M酢酸アンモニウム水溶液(4℃)、同位体で標識した細胞抽出物(4℃)、およびジクロロメタン(-80℃)を含む抽出緩衝液を加えた。細胞破壊、タンパク質変性、および代謝物抽出を、極低温条件下でビーズミリング処理を介して20秒以内に1つの工程で達成した(FastPrep24装置、MP Biomedicals Inc.を使用)。相分離は前述の条件下で遠心分離により行った。あらゆる細胞残屑を完全に除去するため、上部極性相を、前述の条件を用いたスピンろ過により、0.2μmのろ材に対してろ過した。得られたろ液のアリコートを水で希釈し、-80℃で冷凍し、その後凍結乾燥した。
メンブレンを底に張ったインサートを備えた培養皿を使用して接着細胞系を培養した(例えば、トラックエッジ膜、Nunc Inc.)。細胞をサンプリングするために、該膜を切り取り、5個のスチールビーズとジクロロメタンおよびエタノールの2:1混合物(-80℃)とを予め充填した抽出バイアルに移した。抽出のために、1.5 M酢酸アンモニウム水溶液(4℃)および同位体で標識した細胞抽出物(4℃)を加えた。a)に記載したものと同じビーズミリング手順を用いて、細胞と膜を完全に分解した。a)に記載した遠心分離条件を適用してタンパク質、細胞残屑および膜残留物を除去し、試料を上部極性相と下部無極性相とに分離した。上相を集めてから、a)で記載した通りにさらに処理した。
組織をサンプリングするために、該組織を液体窒素の種々の温度で即時冷凍し、その後凍結乾燥した。続いて、3〜5 mgの乾燥組織をジクロロメタンおよびエタノールの2:1混合物(-80℃)で覆った。最後に、1.5 M酢酸アンモニウム水溶液(4℃)および同位体で標識した細胞抽出物(4℃)を加えた。この試料を、b)に記載した通りにさらに処理した。
カンジダ・ユチリス(Candida utilis)(DSMZ sp. 2361として寄託されている)の酵母培養物を、振とうフラスコ内で、アミノ酸を含まないYNB最少培地(Sigma)中のU13C-D-グルコース(10 g/L)上で増殖させた。好気条件を維持するため、該フラスコをオービタルシェーカーを使用して180 rpm、28℃で揺り動かした。細胞の採取は、200 mLの酵母培養物を2分間、1000×gかつ4℃で遠心分離(Falconチューブ)することにより実施した。続いて該細胞を、0.15 M酢酸アンモニウムおよび10 g/L U13C-D-グルコースからなる洗浄用緩衝液と連続した遠心分離を利用して2回洗浄した。得られた細胞ペレットを、7.5 mLの2:1(v/v)ジクロロメタン/エタノール溶液(-80℃)を使用してクエンチした。抽出は、このクエンチ済み試料に1.5 M酢酸アンモニウム水溶液2.5 mLを加えることにより、a)に記載した通りに極低温条件下で実施した。抽出後、試料をa)に記載した条件下で遠心分離してから、U13C-標識代謝物を含む上相を集めて次回使用時まで-80℃で保存した。
リン酸化および/またはカルボキシル化極性代謝物のクロマトグラフ分離のために、超高圧イオン対形成液体クロマトグラフィーIP-UPLCを適用した。溶媒A(脱イオン水)と溶媒B(50%アセトニトリル、50%水(v/v))の間のクロマトグラフ勾配処理を行ったが、一方で一定のカラム流量0.4 mL/分およびカラムオーブン温度45℃を維持した。揮発性添加剤としてトリブチルアミンを両溶離剤に加え、氷酢酸を使用してpH値をpH値6.2に調整することにより、イオン対を形成する(ion pairing)トリブチルアンモニウム陽イオンを生成させた。前記の凍結乾燥試料を低用量の溶離剤Aに溶かした後、前記抽出物1〜20μLを注入した。
負モードのエレクトロスプレー・タンデム質量分析(-ESI-MSMS)を利用して、UPLCにより分離した極性代謝物を評価した。タンデムMSMSはいわゆる計画(scheduled)または選択(selected)多重反応モニタリングモード(sMRM)で操作したが、単位分解能でユニークな質量調整を規定した。個々の代謝物の同位体標識形態および非標識形態は、種々の質量トレース(mass traces)により区別した。
実施例5は、細胞性組織を抽出する際に揮発性酢酸アンモニウムを高濃度で使用したことによる、数種の代謝物の抽出収率の上昇を例示するものである。加えた揮発性緩衝液はその後の凍結乾燥工程中に除去されたが、揮発性が低いリン酸化代謝物は保持された。従って、細胞性組織を高塩濃度緩衝液にさらすと、従来の条件下よりもかなり高い程度まで多数のタンパク質結合代謝物が放出される。この知見は、試料中に少ししか存在しない代謝物にとって特に重要である。さらに、ニコチンアミドの抽出収率の上昇はNADPHの酸化型と還元型の比率に強く影響を与え、このことは細胞または試料における還元当量の入手性を示している(図2)。
実施例6は、凍結乾燥組織の典型的なクロマトグラムを示す。5 mgの乾燥試料は、極低温条件下での抽出前にU13C-標識酵母抽出物を用いて補正(amended)した。図3は、計画的多重反応モニタリングを利用して負モードのESI-MSMSにより測定した70の質量トレース(標識/非標識)のクロマトグラムを示したものである。試料または標識酵母における天然存在度に応じて、質量存在度(mass abundance)には3桁の幅が生じた。オーバービューモード(XIC)の他に、3つの包括的な質量エクストラクト(comprehensive mass extracts)についても図示したが、これらはそれぞれエネルギー代謝、クレブス回路(TCA)、および解糖を表している。
カンジダ・ユチリスsp.(Candida utilis sp.)2361の酵母培養物を、10 g L-1の非標識D-グルコースを使用してYNB培地(Sigma)で増殖させてから、光学密度4.0(600 nm、1 cm光路長)で回収した。クエンチング法によって生じるバイアスを評価するため、該培養物を、
(A)低温溶媒によるクエンチング前に、0.2 μmフィルターに素早く通し、10 g L-1のU13C-標識D-グルコースを含有する150 mMの酢酸アンモニウム緩衝液で洗浄(最大10秒)し、そして
(B)密度勾配遠心分離法を利用して即時クエンチしたが、プロパン1,2-ジオール相は同量のU13C-D-グルコースで補正(amended)した。
Claims (15)
- 極性代謝物を分析するための方法であって、以下:
i)生物学的試料に含まれる細胞の即時破壊を可能にする条件下で、相分離剤と揮発性中性アンモニウム塩とを含む抽出緩衝液を用いて該生物学的試料を抽出する工程、
ii)工程i)で得た抽出物に含まれる極性代謝物をクロマトグラフィーにより分離する工程、および
iii)工程ii)で得た、分離した極性代謝物を分析する工程、
を含み、ここで、工程ii)に先立って、工程i)で得た前記抽出物を、以下の工程:
a)水相のスピン遠心分離、
b)工程a)で得た上清の凍結乾燥、および
c)工程b)で得た凍結乾燥物の、イオン対形成剤を含む分析緩衝液への溶解
により処理する、
上記方法。 - 前記相分離剤がジクロロメタンを含む、請求項1記載の方法。
- 前記揮発性中性アンモニウム塩が酢酸アンモニウム、ギ酸アンモニウム、または炭酸水素アンモニウムである、請求項1または2記載の方法。
- 前記揮発性中性アンモニウム塩の濃度が少なくとも0.5 Mである、請求項1〜3のいずれか1項に記載の方法。
- 前記の工程i)における生物学的試料の抽出を抽出温度-20〜-80℃で実施する、請求項1〜4のいずれか1項に記載の方法。
- 前記イオン対形成剤が、トリブチルアンモニウム陽イオン、トリエチルアンモニウム陽イオン、トリプロピル-アンモニウム陽イオンまたはn-ヘキシルアンモニウム陽イオンである、請求項1〜5のいずれか1項に記載の方法。
- 前記クロマトグラフィーがUPLCまたはHPLCである、請求項1〜6のいずれか1項に記載の方法。
- 前記の工程iii)で得た分離した極性代謝物の分析が質量分析を含む、請求項1〜7のいずれか1項に記載の方法。
- 前記質量分析の方法がESI-MS/MSである、請求項8記載の方法。
- 前記生物学的試料が細胞懸濁液である、請求項1〜9のいずれか1項に記載の方法。
- 前記生物学的試料が膜上で増殖させた接着細胞の培養物である、請求項1〜9のいずれか1項に記載の方法。
- 前記の膜上で増殖させた接着細胞の培養物を、工程i)に先立って、-20〜-80℃の温度を有する請求項1〜4のいずれか1項に記載した抽出緩衝液に該膜を即時移動させることによりクエンチングに供する、請求項11記載の方法。
- 前記の膜上で増殖させた接着細胞の培養物を、工程i)に先立って、液体窒素中に該膜を即時移動させることによりクエンチングに供する、請求項11記載の方法。
- 前記生物学的試料が組織試料である、請求項1〜9のいずれか1項に記載の方法。
- 前記組織試料を、工程i)に先立って、該組織試料を-20〜-80℃の温度まで即時冷凍することにより、または液体窒素を適用することによりクエンチングに供する、請求項14記載の方法。
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