JP2005241424A - Method for preparing sample for use in analysis of proteome - Google Patents
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本発明はプロテオーム解析用のタンパク質試料の調製方法に関し、特に生体由来の試料が高濃度で含有する粘性多糖を迅速かつ効果的に除去し、２次元電気泳動などのプロテオーム解析に好適なタンパク質試料を調製する方法に関する。本発明はまた、プロテオーム解析用のタンパク質試料の調製用試薬に関する。本発明はさらに、プロテオーム解析用の試料の前処理装置および該前処理装置を備えたプロテオーム解析装置に関する。 The present invention relates to a method for preparing a protein sample for proteome analysis. In particular, the present invention relates to a method for preparing a protein sample suitable for proteome analysis such as two-dimensional electrophoresis by quickly and effectively removing viscous polysaccharides contained in biological samples at high concentrations. It relates to a method of preparation. The present invention also relates to a reagent for preparing a protein sample for proteome analysis. The present invention further relates to a sample pretreatment apparatus for proteome analysis and a proteome analysis apparatus including the pretreatment apparatus.
タンパク質研究の分野においては、近年、特定のフェノタイプに関与するタンパク質の探索などを行うために、２次元電気泳動などを用いたタンパク質の網羅的解析（プロテオーム解析）が行われている。プロテオーム解析のための試料の調製には、従来、トライトン X-100（登録商標）、Nonidet P-40またはCHAPS（3-［（3-Cholamidopropyl）dimethylammonio］-1-propanesulfonate）などの界面活性剤を用いてタンパク質を抽出する方法などが用いられてきた。しかし、多糖類を多量に含む試料など、一部の試料については、粘性が高いため、従来の調製法では満足のいく分析結果が得られず、電気泳動を行うことさえもできないものがあった。プロテオーム解析にはより多くのタンパク質をより高い解像度で分離・検出することが重要であるため、プロテオーム解析により適した試料の調製方法の開発が望まれていた。 In the field of protein research, in recent years, comprehensive analysis (proteome analysis) of proteins using two-dimensional electrophoresis or the like has been performed in order to search for proteins involved in specific phenotypes. For the preparation of samples for proteome analysis, surfactants such as Triton X-100 (registered trademark), Nonidet P-40 or CHAPS (3-[(3-Cholamidopropyl) dimethylammonio] -1-propanesulfonate) have been conventionally used. For example, a method for extracting a protein has been used. However, some samples, such as samples containing a large amount of polysaccharides, are so viscous that some conventional preparation methods do not provide satisfactory analysis results and even electrophoresis cannot be performed. . Since it is important to separate and detect more proteins with higher resolution in proteome analysis, development of a sample preparation method suitable for proteome analysis has been desired.
セチルトリメチルアンモニウムブロミド（Cetyltrimethylammonium Bromide）、セチルトリメチルアンモニウムクロリド（Cetyltrimethylammonium Chloride）、セチルピリジニウムブロミド（Cetylpyridinium bromide）、セチルピリジニウムクロリド（Cetylpyridinium chloride）又はセチルジメチルブチルアンモニウムブロミド（Cetyldimethylethylammonium Bromide）などは、４級アンモニウムイオン系界面活性剤に分類され、従来より核酸の精製やタンパク質の変性などに使用されていた。また、キチナーゼの精製に使用できることが報告されているが（非特許文献１参照）、その他の多くの種類のタンパク質の精製にも使用できるかは不明であったため、プロテオーム解析用試料のように多種類のタンパク質を含む試料の調製には使用されていなかった。
本発明は、プロテオーム解析において高い分離度及び検出感度で解析しうるタンパク質試料の調製方法を提供することを課題とする。 An object of the present invention is to provide a method for preparing a protein sample which can be analyzed with high resolution and detection sensitivity in proteome analysis.
本発明者らは、上記課題を解決するために鋭意検討を重ねた。その結果、４級アンモニウムイオンを含む溶液を用いて調製したタンパク質試料を用いることにより、２次元電気泳動の分離度が向上することを見出し、本発明を完成するに至った。 The present inventors have made extensive studies to solve the above problems. As a result, it has been found that the use of a protein sample prepared using a solution containing quaternary ammonium ions improves the resolution of two-dimensional electrophoresis, and the present invention has been completed.
すなわち、本発明は以下の通りである。
（１） 生体試料からプロテオーム解析用のタンパク質試料を調製する方法であって、生体試料または生体試料の抽出液に４級アンモニウムイオン系界面活性剤を含む溶液を加えて沈殿物を形成させ、該沈殿物を分離してタンパク質溶液を得ることを特徴とする方法。
（２） 前記４級アンモニウムイオン系界面活性剤を含む溶液を、前記界面活性剤の最終濃度が０．０５〜０．１％になるように加える、（１）の方法。
（３） 前記界面活性剤がセチルトリメチルアンモニウムブロミド（Cetyltrimethylammonium Bromide）、セチルトリメチルアンモニウムクロリド（Cetyltrimethylammonium
Chloride）、セチルピリジニウムブロミド（Cetylpyridinium bromide）、セチルピリジニウムクロリド（Cetylpyridinium chloride）及びセチルジメチルブチルアンモニウムブロミド（Cetyldimethylethylammonium Bromide）からなる群より選ばれる１又は２種類以上の界面活性剤である、（１）又は（２）の方法。
（４） 前記生体試料が植物由来試料である、（１）〜（３）のいずれかの方法。
（５） ４級アンモニウムイオン系界面活性剤を含むことを特徴とする、プロテオーム解析用試料の調製用試薬。
（６） ４級アンモニウムイオン系界面活性剤を含む沈殿剤供給部、試料導入部、混合部、分離部を含む、プロテオーム解析用試料の前処理装置。
（７） （６）に記載の前処理装置を含むプロテオーム解析装置。
That is, the present invention is as follows.
(1) A method of preparing a protein sample for proteome analysis from a biological sample, comprising adding a solution containing a quaternary ammonium ion surfactant to a biological sample or a biological sample extract to form a precipitate, A method comprising separating a precipitate to obtain a protein solution.
(2) The method according to (1), wherein a solution containing the quaternary ammonium ion surfactant is added so that the final concentration of the surfactant is 0.05 to 0.1%.
(3) The surfactant is cetyltrimethylammonium bromide (Cetyltrimethylammonium Bromide), cetyltrimethylammonium chloride (Cetyltrimethylammonium chloride)
(1) Method (2).
(4) The method according to any one of (1) to (3), wherein the biological sample is a plant-derived sample.
(5) A reagent for preparing a sample for proteome analysis, comprising a quaternary ammonium ion surfactant.
(6) A sample pretreatment apparatus for proteome analysis, including a precipitating agent supply unit including a quaternary ammonium ion surfactant, a sample introduction unit, a mixing unit, and a separation unit.
(7) A proteome analyzer including the pretreatment device according to (6).
本発明の方法によって調製したタンパク質試料を用いることにより、高い分離度及び検出感度でプロテオーム解析を行うことが可能になった。 By using a protein sample prepared by the method of the present invention, it has become possible to perform proteome analysis with high resolution and detection sensitivity.
以下に本発明を詳細に説明する。 The present invention is described in detail below.
本発明の方法は、生体試料からプロテオーム解析用タンパク質試料を調製する方法であって、生体試料または生体試料の抽出液に４級アンモニウムイオン系界面活性剤を含む溶液を加えて沈殿物を形成させ、該沈殿物を分離してタンパク質溶液を得ることを特徴とする方法である。本発明において、プロテオーム解析とは、２次元電気泳動や質量分析などの多種類のタンパク質を含む試料を解析する方法を意味する。また、生体試料としては、生体より得られる試料であれば特に限定されないが、例えば、動物や植物より得られる組織や細胞、微生物の細胞などが挙げられる。これらの中では植物などの多糖類を多く含む試料が、本発明の方法において特に好適に用いることができる。植物由来の試料としては例えば、日本シバ、ヤマノイモ、ラピルスなどが挙げられる。また、粘性多糖類が多い海藻類も好適に用いることができる。 The method of the present invention is a method of preparing a protein sample for proteome analysis from a biological sample, and a solution containing a quaternary ammonium ion surfactant is added to the biological sample or a biological sample extract to form a precipitate. , And separating the precipitate to obtain a protein solution. In the present invention, proteome analysis means a method for analyzing a sample containing many kinds of proteins such as two-dimensional electrophoresis and mass spectrometry. The biological sample is not particularly limited as long as it is a sample obtained from a living body, and examples thereof include tissues and cells obtained from animals and plants, and cells of microorganisms. Among these, a sample containing a large amount of polysaccharides such as plants can be particularly suitably used in the method of the present invention. Examples of plant-derived samples include Japanese shiba, yam, and lapils. Moreover, seaweed with many viscous polysaccharides can also be used suitably.
本発明においては、４級アンモニウムイオン系界面活性剤を含む溶液を生体試料に直接加えてもよいし、生体試料を抽出用バッファーに溶解して抽出液を得た後、該抽出液に４級アンモニウムイオン系界面活性剤を含む溶液を加えもよい。 In the present invention, a solution containing a quaternary ammonium ion surfactant may be added directly to a biological sample, or after the biological sample is dissolved in an extraction buffer to obtain an extract, the quaternary is added to the extract. A solution containing an ammonium ion surfactant may be added.
生体試料に直接４級アンモニウムイオン系界面活性剤を含む溶液を加える場合、タンパク質を安定に保つため、溶液は４級アンモニウムイオン系界面活性剤を含むリン酸緩衝液などの緩衝液を用いることが好ましい。緩衝液には生理的濃度の塩が含まれていてもよい。また、トライトンX-100（登録商標）などの他の界面活性剤が含まれていてもよい。具体的な方法としては、例えば、試験チューブ内などに入れた生体試料に４級アンモニウムイオン系界面活性剤を含む緩衝液を加え、該緩衝液中で生体試料をすりつぶすなどして溶解させる方法を挙げることができる。 When adding a solution containing a quaternary ammonium ion surfactant directly to a biological sample, a buffer solution such as a phosphate buffer containing a quaternary ammonium ion surfactant should be used as the solution in order to keep the protein stable. preferable. The buffer may contain a physiological concentration of salt. In addition, other surfactants such as Triton X-100 (registered trademark) may be included. As a specific method, for example, a buffer solution containing a quaternary ammonium ion surfactant is added to a biological sample placed in a test tube or the like, and the biological sample is ground and dissolved in the buffer solution. Can be mentioned.
一方、生体試料から得た抽出液に４級アンモニウムイオン系界面活性剤を含む溶液を加える場合、まず、リン酸緩衝液、酢酸緩衝液などの抽出用バッファーを用いて生体試料からタンパク質を抽出する。抽出バッファーには生理的濃度の塩が含まれていてもよい。また、トライトンX-100（登録商標）などの他の界面活性剤が含まれていてもよい。抽出は、常法に従って行うことができるが、例えば、試験チューブ内などに入れた生体試料に抽出用バッファーを加えて該試料を溶解し、遠心分離などにより不溶物を除去することにより行うことができる。このようにして得られる抽出液に４級アンモニウムイオン系界面活
性剤を含む溶液を加える。
On the other hand, when adding a solution containing a quaternary ammonium ion surfactant to an extract obtained from a biological sample, first, proteins are extracted from the biological sample using an extraction buffer such as a phosphate buffer or an acetate buffer. . The extraction buffer may contain physiological concentrations of salt. In addition, other surfactants such as Triton X-100 (registered trademark) may be included. Extraction can be performed according to a conventional method, for example, by adding an extraction buffer to a biological sample placed in a test tube or the like, dissolving the sample, and removing insoluble matter by centrifugation or the like. it can. A solution containing a quaternary ammonium ion surfactant is added to the extract thus obtained.
本発明において用いる４級アンモニウムイオン系界面活性剤としては、セチルトリメチルアンモニウムブロミド（Cetyltrimethylammonium Bromide：CTAB）、セチルトリメチルアンモニウムクロリド（Cetyltrimethylammonium Chloride：CTAC）、セチルピリジニウムブロミド（Cetylpyridinium bromide：CPB）、セチルピリジニウムクロリド（Cetylpyridinium chloride：CPC）又はセチルジメチルブチルアンモニウムブロミド（Cetyldimethylethylammonium Bromide：CDEAB）などが挙げられる。これらの４級アンモニウムイオン系界面活性剤は２種類以上を用いてもよい。これらの４級アンモニウムイオン系界面活性剤を含む溶液は、該界面活性剤の最終濃度が０．０１〜０．２％の範囲になるように加えることが好ましく、０．０５〜０．１％になるように加えることがより好ましい。なお、この濃度は試料が含有する粘性多糖の量に影響されるので、適宜調整することも必要である。 Examples of the quaternary ammonium ion surfactant used in the present invention include cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), cetylpyridinium bromide (CPB), and cetylpyridinium chloride. (Cetylpyridinium chloride: CPC) or cetyldimethylethylammonium bromide (CDEAB). Two or more kinds of these quaternary ammonium ion surfactants may be used. The solution containing these quaternary ammonium ion surfactants is preferably added so that the final concentration of the surfactant is in the range of 0.01 to 0.2%, and 0.05 to 0.1%. It is more preferable to add so that it becomes. In addition, since this density | concentration is influenced by the quantity of the viscous polysaccharide which a sample contains, it is also necessary to adjust suitably.
４級アンモニウムイオン系界面活性剤は生体試料中の粘性多糖などと不溶性の複合体を形成する。この複合体を含む沈殿物を遠心分離などにより除去することによってプロテオーム解析用試料を得ることができる。粘性多糖などの不純物を効率よく除くためには、４級アンモニウムイオン系界面活性剤を加えた後、懸濁又は攪拌し、さらに、懸濁及び攪拌の後に、しばらく放置することが好ましい。タンパク質を含む放置する条件は特に制限されないが、低温で放置することが好ましく、例えば、４℃で０．５〜１２時間、放置する条件を例示することができる。放置により、十分に形成された沈殿物を遠心分離などにより除去することにより、プロテオーム解析用タンパク質試料を得ることができる。なお、４級アンモニウムイオン系界面活性剤はDNAなどの核酸を除去する効果も有しているため、本発明の方法によって核酸も同時に除去するができる。本発明の方法によって調製される試料は、粘性が低くプロテオーム解析に好適に用いることができる。 A quaternary ammonium ion-based surfactant forms an insoluble complex with a viscous polysaccharide or the like in a biological sample. A sample for proteome analysis can be obtained by removing the precipitate containing the complex by centrifugation or the like. In order to efficiently remove impurities such as viscous polysaccharides, it is preferable to add a quaternary ammonium ion-based surfactant and then suspend or stir, and then leave it for a while after suspending and stirring. The conditions for allowing the protein to stand are not particularly limited, but it is preferable to leave at a low temperature. For example, the conditions for leaving at 4 ° C. for 0.5 to 12 hours can be exemplified. A protein sample for proteome analysis can be obtained by removing the sufficiently formed precipitate by centrifugation or the like. In addition, since the quaternary ammonium ion surfactant has an effect of removing nucleic acids such as DNA, the nucleic acids can be removed simultaneously by the method of the present invention. The sample prepared by the method of the present invention has a low viscosity and can be suitably used for proteome analysis.
本発明はまた、４級アンモニウムイオン系界面活性剤を含むプロテオーム解析用試料の調製用試薬を提供する。本発明の試薬は、さらに、タンパク質抽出液を含むものであってもよい。このような試薬としては、例えば、４級アンモニウムイオン系界面活性剤溶液およびタンパク質抽出用リン酸緩衝液を含むプロテオーム解析用試料の調製用試薬等が挙げられる。 The present invention also provides a reagent for preparing a sample for proteome analysis containing a quaternary ammonium ion surfactant. The reagent of the present invention may further contain a protein extract. Examples of such a reagent include a reagent for preparing a sample for proteome analysis including a quaternary ammonium ion surfactant solution and a phosphate buffer for protein extraction.
本発明はまた、プロテオーム解析用試料の前処理装置を提供する。本発明の前処理装置は、試料導入部、４級アンモニウムイオン系界面活性剤を含む沈殿剤供給部、混合部、分離部を含む、前処理装置である。 The present invention also provides a sample pretreatment apparatus for proteome analysis. The pretreatment apparatus of the present invention is a pretreatment apparatus including a sample introduction part, a precipitant supply part containing a quaternary ammonium ion surfactant, a mixing part, and a separation part.
このような前処理装置としては、例えば、図４の「前処理部」に示すような装置を挙げることができる。この装置においては、試料は試料供給部（２０３）からインジェクタ（２０４）により注入され、溶媒供給部（２０１）から微量ポンプ（２０２）を通って供給される溶媒によりミキサー部（２０７）に送られる。沈殿剤は沈殿剤供給部（２０５）から微量ポンプ（２０６）によりミキサー部（２０７）に送られる。ミキサー部において試料と沈殿剤が混合され、反応部（２０８）において沈殿形成反応が進行する。反応部（２０９）において充分反応させた後、混合液は分離部（２１０）に送られ、沈殿物とタンパク質溶液に分離される。微量ポンプ、インジェクタ、ミキサーなどは通常のものを組み込むことができる。分離部としては、ろ過装置や遠心分離装置などが挙げられるが、試料の粘性が高い場合は遠心分離装置が好ましい。 As such a pre-processing apparatus, for example, an apparatus as shown in “Pre-processing section” in FIG. 4 can be cited. In this apparatus, the sample is injected from the sample supply unit (203) by the injector (204), and sent to the mixer unit (207) by the solvent supplied from the solvent supply unit (201) through the micro pump (202). . The precipitating agent is sent from the precipitating agent supply unit (205) to the mixer unit (207) by the micro pump (206). The sample and the precipitating agent are mixed in the mixer section, and the precipitation forming reaction proceeds in the reaction section (208). After sufficiently reacting in the reaction section (209), the mixed solution is sent to the separation section (210) to be separated into a precipitate and a protein solution. A micro pump, an injector, a mixer, etc. can incorporate a normal thing. Examples of the separation unit include a filtration device and a centrifuge, but a centrifuge is preferable when the viscosity of the sample is high.
本発明は、さらに、このような前処理装置を含むプロテオーム解析装置であってもよい。例えば、図４に示すような液体クロマトグラフィー／質量分析装置（LC-MASS）装置を挙げることができる。この装置においては、前処理部（２１１）から修飾反応部（２１２
）及び酵素反応部（２１３）を通ってインジェクタ（２２０）により供給される酵素分解ペプチドが、溶離液供給部（２１５及び２１６）から微量ポンプ（２１７及び２１８）により送られミキサー部（２１９）内で混合された溶離液によって、カラム（２２１）内で展開・分離され、質量分析機（２２２）内で質量分析にかけられ、さらに、データ処理部（２２３）で解析される。カラムとしては通常の質量分析用カラムを用いることができ、データ処理部としては、通常のコンピューターなどを用いることができる。
The present invention may further be a proteome analysis device including such a preprocessing device. An example is a liquid chromatography / mass spectrometer (LC-MASS) apparatus as shown in FIG. In this apparatus, the pre-processing unit (211) to the modification reaction unit (212)
) And the enzyme reaction unit (213), and the enzyme-degraded peptide supplied by the injector (220) is sent from the eluent supply unit (215 and 216) by the micropumps (217 and 218) and in the mixer unit (219). In the column (221), the eluent mixed in (1) is developed and separated, subjected to mass spectrometry in the mass spectrometer (222), and further analyzed by the data processing unit (223). A normal mass analysis column can be used as the column, and a normal computer or the like can be used as the data processing unit.
以下、本発明を実施例によりさらに具体的に説明する。但し、本発明はこれらの実施例に限定されない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
［実施例１］SDS電気泳動による評価
生体試料として日本シバを用いた。該試料に３倍量のリン酸緩衝液を加えて溶解し、遠心分離を行い、不溶物を除去してタンパク質抽出液を得た。該抽出液100μＬに5μＬの２％CPC（塩化エキサデシルピリジニウム一水和物；ナカライテスク製）を添加して攪拌した。攪拌後、４℃で一晩放置し、沈殿物を形成させた。12,000rpm、4℃、5分間の遠心分離により沈殿物を除去し、上清を回収してタンパク質試料を得た。該試料10μＬをSDS-ポリアクリルアミド電気泳動により解析した（レーン３）。対照として、CPC未処理サンプル（レーン２）及び2D Clean Up Kit (Amersham Biosciences社) （レーン４）を用いて調製したサンプルを同時に電気泳動により解析した。結果を図１に示す。レーン１及び５は分子量マーカー（ウシ血清アルブミン：６６ｋＤａ，カルボニックアンヒドラーゼ：２９ｋＤａ，トリプシンインヒビター：２０ｋＤａ，ニワトリリゾチーム：１４ｋＤａ）を示す。
[Example 1] Evaluation by SDS electrophoresis Nippon Shiba was used as a biological sample. The sample was dissolved by adding 3 times the amount of phosphate buffer, centrifuged, and insoluble matter was removed to obtain a protein extract. To 100 μL of the extract, 5 μL of 2% CPC (exadecylpyridinium chloride monohydrate; manufactured by Nacalai Tesque) was added and stirred. After stirring, the mixture was left overnight at 4 ° C. to form a precipitate. The precipitate was removed by centrifugation at 12,000 rpm at 4 ° C. for 5 minutes, and the supernatant was recovered to obtain a protein sample. 10 μL of the sample was analyzed by SDS-polyacrylamide electrophoresis (lane 3). As controls, a CPC-untreated sample (lane 2) and a sample prepared using 2D Clean Up Kit (Amersham Biosciences) (lane 4) were simultaneously analyzed by electrophoresis. The results are shown in FIG.
CPC未処理サンプルに比べて高分子のバンドがより多く検出できた。これにより、CPCを用いてタンパク質試料を調製することにより電気泳動の分離度及び解像度が著しく向上することがわかった。なお、2D Clean Up Kitによって調製された試料についても高分子バンドは検出されたが、バックグラウンドが上昇しバンドのコントラストが低下した。 More polymer bands were detected compared to the CPC untreated sample. As a result, it was found that the resolution and resolution of electrophoresis are remarkably improved by preparing a protein sample using CPC. In the sample prepared with the 2D Clean Up Kit, a polymer band was detected, but the background increased and the band contrast decreased.
［実施例２］２次元電気泳動による評価（１）
生体試料として粘性の非常に高いヤマノイモのムカゴ（気根）を用いた。該試料に3倍量の0.1M酢酸緩衝液（pH4.0）を加えて溶解させた。遠心分離を行い、不溶物を除去してタンパク質抽出液を得た。該抽出液100μＬに２％CPCを１／２０量（５μＬ）加えて攪拌し、４℃で一晩放置後、遠心分離により上清画分を得た。この上清画分５μＬに膨潤緩衝液（８Ｍ尿素、２％ＣＨＡＰＳ，２％ＩＰＧ緩衝液、０．３％ＤＴＴ）１２５μＬを加え、同溶液にてＩＰＧドライストリップ（７ｃｍ、ｐＨ３−１０；アマシャムファルマシア製）を一晩膨潤させた後、２次元電気泳動を行った。対照として、CPC未処理のサンプルについても電気泳動を行った。なお、電気泳動は、１次元にはＩＰＧ−ＩＥＦ ＴｙｐｅＣ（アナテック製）、２次元目にはＡＥ６５００（アトー製）を用いた。結果を図２に示す。
[Example 2] Evaluation by two-dimensional electrophoresis (1)
As a biological sample, a wild yam mugago (air root) was used. Three times the amount of 0.1 M acetate buffer (pH 4.0) was added to the sample and dissolved. Centrifugation was performed to remove insoluble matter, and a protein extract was obtained. 1/20 volume (5 μL) of 2% CPC was added to 100 μL of the extract, and the mixture was stirred and allowed to stand overnight at 4 ° C., followed by centrifugation to obtain a supernatant fraction. 125 μL of swelling buffer (8M urea, 2% CHAPS, 2% IPG buffer, 0.3% DTT) was added to 5 μL of this supernatant fraction, and IPG dry strip (7 cm, pH 3-10; Amersham Pharmacia) was added in the same solution. The product was swollen overnight, and then two-dimensional electrophoresis was performed. As a control, electrophoresis was also performed on a sample not treated with CPC. For electrophoresis, IPG-IEF TypeC (manufactured by Anatech) was used in the first dimension, and AE6500 (manufactured by Ato) was used in the second dimension. The results are shown in FIG.
CPC未処理の試料（B）では、ほとんどタンパク質のスポットが検出されなかったが、CPC処理を行った試料（A）においては、多数のタンパク質のスポットが検出された。 In the sample not treated with CPC (B), almost no protein spots were detected, but in the sample subjected to CPC treatment (A), many protein spots were detected.
［実施例３］２次元電気泳動による評価（２）
生体試料としてアピオス（アメリカ大陸原産のイモ）を用いた。該試料に3倍量のリン酸緩衝液（pH7.0）を加えて溶解し、遠心分離を行い、不溶物を除去してタンパク質抽出液を得た。該抽出液100μＬに２％CPCを１／２０量（５μＬ）加えて攪拌し、４℃で一晩放置後、遠心分離により上清画分を得た。この上清画分５μＬに膨潤緩衝液（８Ｍ尿素、２％ＣＨＡＰＳ，２％ＩＰＧ緩衝液、０．３％ＤＴＴ）１２５μＬを加え、同溶液にてＩ
ＰＧドライストリップ（７ｃｍ、ｐＨ３−１０；アマシャムファルマシア製）を一晩膨潤させた後、２次元電気泳動を行った（A）。対照として、CPC未処理のサンプルについても電気泳動を行った（B）。結果を図３に示す。CPC処理の方がCPC未処理に比べてタンパク質スポットの数が顕著に多いことがわかる。以上の結果から、CPC処理を行うことにより、２次元電気泳動に好適な試料が得られることがわかった。なお、実施例においてはCPCを用いた例を示したが、CTABなどの他の4級アンモニウムイオン系界面活性剤を用いることによっても、同様に２次元電気泳動に好適な試料が得られることが本発明者によって確認されている。
[Example 3] Evaluation by two-dimensional electrophoresis (2)
Apios (a potato native to the American continent) was used as a biological sample. Three times the amount of phosphate buffer (pH 7.0) was added to the sample for dissolution, and centrifugation was performed to remove insoluble matters to obtain a protein extract. 1/20 volume (5 μL) of 2% CPC was added to 100 μL of the extract, and the mixture was stirred and allowed to stand overnight at 4 ° C., followed by centrifugation to obtain a supernatant fraction. To 5 μL of this supernatant fraction, 125 μL of swelling buffer (8 M urea, 2% CHAPS, 2% IPG buffer, 0.3% DTT) was added, and I
PG dry strip (7 cm, pH 3-10; manufactured by Amersham Pharmacia) was swollen overnight and then subjected to two-dimensional electrophoresis (A). As a control, electrophoresis was also performed on a sample not treated with CPC (B). The results are shown in FIG. It can be seen that the number of protein spots is significantly higher in the CPC treatment than in the non-CPC treatment. From the above results, it was found that a sample suitable for two-dimensional electrophoresis can be obtained by performing CPC treatment. In addition, although the example using CPC was shown in the Examples, a sample suitable for two-dimensional electrophoresis can be obtained similarly by using other quaternary ammonium ion surfactants such as CTAB. Confirmed by the inventor.
本発明により、高分離度及び高感度でのプロテオーム解析が可能になった。 The present invention has enabled proteomic analysis with high resolution and high sensitivity.
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| WO2008051178A3 (en) * | 2006-04-12 | 2009-05-07 | Savient Pharmaceuticals Inc | Purification of proteins with cationic surfactant |
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