MHが「ドイツ学術振興」（DFG）からの支援を認めるものです。著者 寄付：MHが調査を設計し、KT、JSとMTは、研究を行い、データを分析し、KTとMHが論文を書いた。

著者らは、競合する経済的利害関係を宣言していません。

安定同位体標識を用いて、異なる定量的プロテオミクス研究は、過去数年に発表されている。これらの実験では、通常、2つの異なるサンプルは、1つのサンプルが安定同位体で標識された、比較される。その後、二つの試料からのタンパク質またはペプチドは、等しい比率で組み合わされ、一緒になって、さらに48を処理した。このような研究は、多くの場合、アップまたはダウンレ?...

植物や藻類のチラコイド膜での光合成のプロセスは、線状および環状モードで機能することができます。リニア電子流（LEF）の間に光化学I（PSI）、光化学系II（PSII）およびシトクロムB 6 / Fは、最終的には 、NADPHとATP 2の生成につながる、NADP + 1を水から電子を移動。対照的に、状態2 3 4及び嫌気的条件等の多様な環境条件下で誘導されることが知られている環状の電子流（CEF）は、バック電子伝達鎖に電子を注入することによって酸化PSIの再減少をもたらす。このプロセスは、チトクロームB 6 / F複合体1の間質側またはプラストキノンプール5のいずれかで行われるとATPを生成しますが、無NADPH 2することができます。 提示されたプロトコルの目的は、質量分析（MS）ベースのm個を実証することである（遺伝的に異なる株と比較することによって例示される）、さまざまな条件の下でこれらの複合体の組成物への洞察を得るためにクラミドモナスのチラコイド膜での多タンパク質複合体の比較、定量分析のためのethod。このアプローチは、寺島らによる刊行物に適用された。2012年にはC言語でのCEFののCa 2 +依存性の調節を示すクラミドモナスは、タンパク質、CAS、ANR1、およびPGRL1 6を含む多タンパク質複合体により媒介される。手順は、比較的重いことにより、標識窒素（15 N）を有する二つの株のいずれかを利用して二つ遺伝的に異なる菌株におけるCEF-supercomplexの組成を分析することによって説明される。簡単に説明すると、プロトコルは、界面活性剤可溶化し、ショ糖密度勾配における光合成複合体の分画、続いてチラコイド膜の調製を含む。勾配の分画後、FRACを選択した二つの株のtionsを混合し、等しい体積に基づいてゲル内消化およびその後の定量的MS分析に続いてSDS-PAGEによって分離されている。 上述したように、CEFは異なる環境条件の下で誘導され、2010年からの出版物は、機能的なCEF-supercomplex Cの状態2ロックされたセルからの隔離を示しています超遠心分離中にショ糖密度勾配上に可溶化されたチラコイド膜を分離することによって行われた、07 ラインハーディ 。岩井ら 7とは異なり、提示されたプロトコルは、C.成長嫌気から、CEF-supercomplexの単離を記載代替手順に従うことによって、 クラミドモナスの文化。これは、チラコイド分離プロトコルにおける変化ならびに可溶化工程及び超遠心分離によるタンパク質複合体の分離に関する差異を含む。現在のプロトコルでは、チラコイド膜バッファは、岩井らによるチラコイド調製のために使用しながら、チュアとBennoun 8によって発表された手順を適用することによって単離される文献 09記載のように、25mMのMES、0.33 Mスクロース、5mMのMgCl 2、1.5mMのNaCl液（pH6.5）を含有した。ここで説明可溶化法が0.9％の界面活性剤の使用に依存しながら可溶化は、岩井および共同研究者の場合には、氷上で30分間、0.7から0.8パーセントの界面活性剤（n-トリデシル-β-D-マルトシド）を用いて行った（nは-ドデシル-β-D-マルトシド（DM-β））と、氷上で20分間だけ実行される。両グループは、それぞれの洗剤で可溶化にミリリットルあたりのクロロフィル0.8mgのを使用していました。このプロトコルの作者が範囲の濃度を使用し、一方、可溶化したチラコイド膜からの光合成複合体を分離するための岩井ら 、0.1から1.3 mのショ糖濃度を適用0.4〜M.最後の差から比べても低い遠心分離速度は、あるEAへrlier出版。 ショ糖密度勾配分画に続いて非イオン性界面活性剤とのチラコイド膜の可溶化は、すでに1980年代から、タンパク質の代謝標識の適用はプロテオミクス分野で普及の方法で、今日7、9月14日まで、至るまで数多くの研究に適用されている。記載されたアプローチは、質量に至る全てのアミノ酸に組み込まれている15 N NH 4 Cl を 、の形の唯一の窒素源として重窒素の存在下で培養することにより、2つの比較した株のいずれかの15 N代謝標識を適用するペプチドのアミノ酸配列に応じてシフトする。 1 MSラン内の14 Nおよび15 Nの混合物を分析する場合、この質量シフトは、各ペプチドの試料の起源を決定するために使用することができ、ペプチドの相対存在量は、相当の相対存在量を表す計算することができる。タンパク質15を ING。 C上の多数の定量的プロテオミクス研究クラミドモナスは、実験条件（ 例えば 、栄養16〜19によるプロテオームの変化や光ストレス20,21）間のプロテオームの変化を分析するために、タンパク質の定義された量を比較した、利用可能です。これらの研究と比較して、現在提示アプローチにおいて試料の等容量を一緒にし、分析する。このセットアップでは、グラデーション内のタンパク質の移行挙動を研究するため、さらに調査した株に対して異なる複合体の組成を分析することができます。 このメソッドは、主に三つのタンパク質に集中して説明する。第一候補は、C.中の光馴化に関与することが示された葉緑体に局在するカルシウムセンサータンパク質のCASで、 クラミドモナス 22。カルシウムは、重要なシグナルであると考えられている最終的に遺伝子発現と細胞生理学23の変化につながるため、様々な生物的および非生物的ストレスに活性化され、それが葉緑体は、CASタンパク質22,24,25を介したシグナル伝達+携帯のCa 2に貢献するかもしれないことが提案された経路のためのイオンをING。第二のタンパク質はANR1（嫌気応答1 6）、Cで無酸素の成長条件下で誘導されることが示されたタンパク質であるクラミドモナス 26。とりわけ、CASならびにANR1はCEF-supercomplexのサブユニットとして同定され、また、逆遺伝学的ア ​​プローチを用いて、それが両方のタンパク質が、このタンパク質複合体の機能的なサブユニットとしての役割を支持し、 インビボ 6 に CEFに機能的に寄与することが実証された。第3のタンパク質は、 クラミドモナス 4,27においてだけでなく、 シロイヌナズナ 5,28にCEFに関与することが示されており、また、IDだったチラコイド蛋白PGR5様1（PGRL1）、ある岩井らの作品にentified。7 ΔPSI29株（対）対野生型（WT）、PSAB遺伝子の欠失を示す、またの一部であるサブユニットIは本質的な光化学系をコードする：このアプローチは、2つの異なる実験の結果を示すことによって提示されるCEF-supercomplex及びWT対A pgrl1ノックアウト株は4。これらの実験のそれぞれについて、15 Nおよび14 N標識株は、比較されている間、CEF-supercomplexの定量的組成物。

<table border="0" cellpadding="0" cellspacing="0" width="1135">
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			<td height="21" style="height:21px;width:392px;">Chemicals</td>
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			<td height="42" style="height:42px;width:392px;">Acetic acid</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
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			webiste: http://www.applichem.com/home/</td>
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			<td></td>
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			<td height="63" style="height:63px;">Acetonitrile Optigrade f&#252;r LC-MS</td>
			<td style="width:427px;">Diagonal 
			website: https://www.diagonal.de/</td>
			<td style="width:119px;">9340</td>
			<td style="width:199px;">harmful, work with gloves 
			see protocol text for further precautions</td>
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			<td height="45" style="height:45px;">Ammonium chloride 15N</td>
			<td style="width:427px;">Cambridge Isotope Laboratories 
			website: http://www.isotope.com/cil/index.cfm</td>
			<td style="width:119px;">39466-62-1</td>
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			<td style="width:427px;">AppliChem 
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			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
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			<td height="42" style="height:42px;">Ammonium sulfate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3598</td>
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			<td style="width:427px;">Fisher Scientific 
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			<td>10041653</td>
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			<td height="42" style="height:42px;">n-Dodecyl-&#946;-D-maltoside</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
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			<td height="42" style="height:42px;">EDTA</td>
			<td style="width:427px;">AppliChem 
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			<td style="width:119px;">A2937</td>
			<td></td>
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			<td height="42" style="height:42px;">Formic acid&#160;&#160;</td>
			<td style="width:427px;">AppliChem 
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			<td style="width:119px;">A3858</td>
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			<td style="width:427px;">AppliChem 
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			<td style="width:119px;">A3724</td>
			<td></td>
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			<td height="42" style="height:42px;">Magnesium chloride</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td>A4425</td>
			<td></td>
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			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2954</td>
			<td></td>
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			<td height="42" style="height:42px;">Phosphorous acid</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
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			<td></td>
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			<td height="42" style="height:42px;">Di-Potassium hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1042</td>
			<td></td>
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			<td height="42" style="height:42px;">Potassium di-hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
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			<td></td>
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			<td height="42" style="height:42px;">Sodium hydroxide</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1551</td>
			<td></td>
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			<td style="width:119px;">A1125</td>
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			<td></td>
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			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
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			<td></td>
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			<td height="42" style="height:42px;">Trypsin (sequencing grade modified) and Trypsin buffer</td>
			<td style="width:427px;">Promega 
			website: http://www.promega.de/</td>
			<td style="width:119px;">V5111</td>
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			<td style="width:427px;">Glas-Col 
			website: http://www.glascol.com/product/subproduct/id/75</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:392px;">Centrifuge tubes (14 x 89 mm)&#160; 
			for preparation of takahashi style gradients</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;">331372</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Centrifuge tubes 25 x 89 mm 
			for preparation of thylakoid isolation gradients&#160;</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
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			<td></td>
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			<td height="42" style="height:42px;width:392px;">Coulter Avanti Centrifuge J-20 XP</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;">Fuchs-Rosenthal cell couting chamber</td>
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			<td style="width:427px;">Fisherbrand 
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			<td style="width:119px;">105252220</td>
			<td></td>
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			<td height="42" style="height:42px;width:392px;">Ultracentrifuge (Optima XPN-80 Ultracentrifuge)</td>
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			<td height="42" style="height:42px;width:392px;">Antibodies&#160;</td>
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			<td style="width:119px;"></td>
			<td></td>
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</table>

a new approach for the comparative analysis of multiprotein complexes based on 15n metabolic labeling and quantitative mass spectrometry

導入されたプロトコルは、異なる条件下での複雑な組成物への洞察を明らかにすることによって、チラコイド膜内の多タンパク質複合体の分析のためのツールを提供しています。このプロトコルではアプローチは、遺伝的に異なる株から単離されたコナミドリムシ環状電子流（CEF）に関与するタンパク質複合体の組成を比較することによって実証される。手順は、差動代謝標識（14 N / 15 N）に基づいて、ショ糖密度勾配遠心分離、SDS-PAGE、免疫検出および比較、定量的な質量分析（MS）による多タンパク質複合体へのそれらの分離を行い、チラコイド膜の単離を含む株を分析した。洗剤可溶化したチラコイド膜は、等しいクロロフィル濃度のショ糖密度勾配にロードされます。超遠心分離後、勾配は、質量spectrometによって分析される画分に分離されるRY同体積に基づく。このアプローチは、さらに勾配画内の組成の調査と、特にANR1、CAS、およびPGRL1に焦点を当て、異なるタンパク質の移行挙動を分析することができます。さらに、この方法は、（イムノブロットの結果を確認することにより、さらに、以前の研究からの知見を支持して、以前にCEF-supercomplex例えばPGRL1、FNRなどの一部であると記載されたタンパク質の同定およびPSI-依存性遊走を示しており、 CYT F）。注目すべきことに、このアプローチは、このプロトコルを採用することができ、 例えば、異なる環境条件から単離された多タンパク質複合体組成物の比較分析のために使用される質問の広い範囲に対処するために適用可能である。

導入された定量的プロテオミクスのアプローチは、遺伝的に異なる温度でのCEF-supercomplexコンポーネントの比較分析によって実証チラコイド膜内の多タンパク質複合体の組成物を特徴づけることを目的クラミドモナス株 。 。メソッドが正常寺島らにより適用された6記載の洗剤可溶化に続いて、嫌気性成長した培養物からのチラコイド膜の単離を含む。次い?...

Watch this Scientific Journal Video about A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry at JoVE.com

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

記載された比較、定量的プロテオミクスアプローチは、異なる条件下で多タンパク質複合体の組成物への洞察を得ることを目的と遺伝的に異なる菌株を比較することによって実証される。定量分析のためにショ糖密度勾配とは異なる画分の等し​​い体積を質量分析によって混合し、分析する。

に基づいた多タンパク質複合体の比較分析のための新たなアプローチ<sup&gt; 15</sup&gt; N代謝標識および定量的質量分析

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

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Research

JoVE Journal

Biology

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

12.1K ビュー.  University of Münster. 記載された比較、定量的プロテオミクスアプローチは、異なる条件下で多タンパク質複合体の組成物への洞察を得ることを目的と遺伝的に異なる菌株を比較することによって実証される。定量分析のためにショ糖密度勾配とは異なる画分の等し​​い体積を質量分析によって混合し、分析する。 

ビデオ: A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDIサンプル調製：超薄層法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

生物学

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

エレクトロスプレーイオン化質量分析法を用いて酵母Lipidomeの定量評価

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

構造質量分析法によって大きなタンパク質複合体の分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

細胞溶解物からの多タンパク質複合体の解析のための青いネイティブポリアクリルアミドゲル電気泳動（BN - PAGE）

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

PAK2活性化のアミド水素/重水素交換とMALDI - TOF質量分析

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

に基づいて、タンパク質 - タンパク質相互作用の同定のためのプロトコル<sup&gt; 15</sup&gt; Nの代謝標識、免疫沈降法、定量的質量分析法と親和性調節

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

ChroPアプローチは、クロマチンの遺伝子座特異的プロテオームの風景を分析するために、チップと質量分析を組み合わせ

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

グレースケール解析を用いたマウスの抜け毛の定量分析のための新たな手法

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

部位特異的タンパク質のリジンのアセチル化のサクシニル化化学量論組成データに依存しない取得質量分析法による定量

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...