この作品は、NCI臨床プロテオミクス技術評価センター（CPTAC）助成金（＃U24 CA126476）だけでなく、娯楽産業財団（EIF）とEIFの女性の癌研究基金から乳がんのバイオマーカー探索コンソーシアムへの助成金によって賄われて、とされケック財団、カナリー財団、およびポールG.アレンファミリー財団からの寛大な贈り物。

実験方法： アッセイは、合成ペプチドおよび抗ペプチド抗体を必要とします。選択されたペプチドは、目的のタンパク質に固有のものでなければ、8〜22アミノ酸を含む、および既知の翻訳後修飾を持たせるべきではありません。メチオニン残基は一般的に回避し、二塩基性アミノ酸（例えば（株）、KR、RR）を含むペプチドは望ましくないされています。この手法の場合、それは、ペプチドのC末端（すなわち、KまたはRがラベル）で（13 Cおよび15 N）重い標識アミノ酸を組み込んだ、内部標準として安定同位体標識されたペプチドを使用するのが一般的です。 次のプロトコルは、Epitomics社（バーリンゲーム、カリフォルニア州）とニューイングランドペプチド（ガードナー、MA）から合成ペプチドから得られた抗ペプチド抗体を用いて、マウスのタンパク質オステオポンチンからペプチドGDSLAYGLRを測定するために開発されたアッセイを説明します。 1）ペプチド3の濃縮）質量分析法による分析、複雑なタンパク質混合物、2のトリプシン消化を）：プロトコルは、主に3つの手順（図1）で構成されています。それは、マウスオステオポンチン蛋白質を添加したヒト血漿サンプルで展示されます。 1。トリプシン酵素消化およびクリーンアップ<ol><li>氷上で10μLきちんとした血漿のアリコートを解凍。 </li><li> BCAアッセイによる総タンパク質濃度を決定し、任意の懸濁物質を除去するためにサンプルを遠心してください。 </li><li>その蓄積管から1000μLディープウェルプレートとピアス可能なフィルムでカバーにピペット10μL分注し。 </li><li>各サンプルに新鮮9M尿素/ 30mMのジチオスレイトール（DTT）（最終濃度6Mの尿素/ 20mMのDTT）の20μLを追加。 </li><li> 37℃で30分間インキュベート℃に</li><li>各サンプルに新鮮な500mMのヨードアセトアミド（最終IAM 50mm）を3μLを加える。 </li><li> 、暗所、室温で30分間インキュベートする。 </li><li> 100mMのトリス（pH 8）（〜0.6Mに希釈するの尿素）257μLを加える。 </li><li>トリプシンストック溶液（;：基板の比1:50酵素のための1μg/μL）を10μLを加える。 </li><li>インキュベート37 ° Cで一晩（12〜16時間）。 </li><li>きちんとしたギ酸（終濃度1％）の3μLを加える。 </li><li>安定同位体の標準を（多重化アッセイを行う場合は、複数の規格が追加され、通常、これは標準的な同位体標識されたペプチドの10μLを含む50〜100 fmolの程度）に追加します。 </li><li> 80％のアセトニトリル、廃棄フロースルーの0.1％ギ酸500μLを加えオアシスカートリッジのプレートを洗浄してください。この3回繰り返します。 </li><li>水中0.1％ギ酸500μLを加えることによってカートリッジのプレートを平衡化し、フロースルーを捨てる。この4回繰り返します。 </li><li>カートリッジのプレートにダイジェストのサンプルをロードし、流れが非常に遅いですので、真空を調整する。 </li><li>水中0.1％ギ酸500μLで洗浄し、フロースルーを捨てます。この3回繰り返します。 </li><li> 1000μlのディープウェルプレート（フロースルーを破棄しない）に80％アセトニトリルの0.1％ギ酸の2 × 500μLを添加することにより溶出するペプチド。 </li><li>乾燥するまで（またはspeedvac）溶出液を凍結乾燥。 （凍結乾燥が好ましい方法です） </li><li>再構成するには、50μLPBS +0.03％CHAPSを添加することによりペプチドを乾燥させた。 </li></ol> 2。ペプチドのイムノアフィニティー濃縮<ol><li>標準的なキングフィッシャー96ウェルプレートにサンプルを移す。 </li><li> 1μgの抗体とターゲットごとに1.5μLプロテイン- G被覆磁性ビーズを（それが前の加算にビーズに抗体を架橋するためのオプションです）を追加します。ビーズをよく振ったり、ボルテックスで中断されていることを確認します。 </li><li>箔でプレートをカバーしています。 </li><li>穏やかにビーズが中断されるように転倒して（12〜16時間）一晩インキュベートする。 </li><li>箔の表面から液体を除去するために5秒間に32 × gでプレートを遠心する。 </li><li>ホイルカバーを取り外します。 </li><li>ビーズの洗浄および溶出は、手動または自動化された方法で行うことができます。この手順では、カワセミのプラットフォーム上で自動化手順を説明します。 </li><li> 200μLのPBS +0.03％CHAPS（洗浄あたり1分）でビーズを2回洗浄する。 </li><li> PBS 200μLの1:10希釈液+ 0.03％CHAPS（1分）でビーズには1回洗浄する。 </li><li>ペプチドは、5％酢酸+ 0.03％CHAPS、25 uLのに溶出されます。 </li><li>磁石で溶出プレートを配置し、残りのビーズを転送しないように注意しながら、96ウェルプレートに溶出液を移す。 </li><li>シーリングマットとプレートをカバーしています。 </li><li>溶出液を含有するプレートは、分析のためのトリプル四重質量分析計に配信されます。 </li></ol> 3。マルチプルリアクションモニタリングによる分析 - 質量分析<ol><li> SRM / MRM分析のための遷移は0.5μL/ minで質量分析計に30％アセトニトリル％ギ酸でペプチド標準のマイクロリットルのソリューションごとに1ピコモルを導入することで、選択して最適化することができます。安定した噴霧が得られれば、MS / MSスペクトルを収集する。 </li><li>遷移は、THREを識別することにより、MS / MSスペクトルから選択されています電子ノイズの少ないスペクトルの領域において豊富なフラグメントイオン。多価ペプチドイオンの場合、y -イオンのフラグメントは、m /でdetected Z&gt;前駆体のm / zは、通常、SRM / MRMアッセイの開発に最適です。 図2に示す476.3 MS / MSスペクトルと、選択された遷移ions&gt; 508.3、579.3 、ペプチドGDSLAYGLR用692.4（重い安定同位体標準481.3の遷移が&gt; 518.3、589.3、702.4示されていない）。 </li><li>使用される質量分析装置の製造元とモデルに応じて、各遷移に合わせて最適化できる複数のパラメータがあります。ここでは、衝突エネルギーをランピングし、信号のレベルを監視することによって、各選択された遷移の衝突エネルギーを最適化。 25の衝突エネルギーは、それぞれの移行のために使用されていました。 </li><li>次のように簡潔に、SRM / MRM分析のための一般的な構成は、次のとおりです。移動相（）0.1％ギ酸、（B）90％アセトニトリル/ 0.1％ギ酸、0.3 × 5mmのC18トラップカラム、内径75μmx 15 cmのC18分析カラム（Reprosil - PUR C18 AQ、120 °の細孔）。注入量は10μLで、サンプルは、総流量3μL/分で3％Bで5分間、ロードされ、3からの直線勾配により溶出される - 300で45％のB - 10分400 NL /分。 4000 QTRAP（ABSCIEX、フォスターシティー、CA）上の条件は、スプレー電圧2.3kV、イオン源温度150℃、12 GS1、およびカーテンガス15人。 </li><li>サンプルは、サンプルの10μLを注入することにより、SRM / MRMで分析される。ピーク面積は、プログラムのスカイラインを使って光と重いペプチドのために統合されています。14はこのケースでは、バックグラウンドノイズのない最も豊富なトランジションを使用して各サンプル中の非標識/標識ペプチドのピーク面積比（PAR）、Y5の移行を計算する（光ペプチド476.3&gt; 579.3、重い標準ペプチド481.3&gt; 589.3）。 </li></ol> 4。代表的な結果： 測定されたピーク面積比（スパイク重い同位体標識されたペプチドに光内因性ペプチドの相対的な）ターゲットペプチドの定量的尺度を提供する。 図3は、SISCAPA -濃縮試料中の軽鎖および重ペプチドの例のクロマトグラムを示しています。同じ時間と複数の遷移で光と重いペプチドの溶出が身元を確認するために、各ペプチドのために監視することができることに注意してください。 <img src="/files/ftp_upload/2812/2812fig1.jpg" alt="図1"/> 図1。SISCAPAプロセスの概要図。複雑なタンパク質混合物は、ペプチドに消化される。標的ペプチドの分析対象物（内因性の分析対象とスパイク安定同位体標識内部標準）は、プロテインG -コートされた磁性粒子に固定化された抗ペプチド抗体を用いて濃縮される。単離後、標的ペプチドは磁性粒子から溶出され、内部標準に対する相対定量用質量分析法により分析した。 <img src="/files/ftp_upload/2812/2812fig2.jpg" alt="図2"/> 図2。SRM / MRMトランジションのための3つのフラグメントの選択を示すペプチドGDSLAYGLRのMS / MSスペクトル。 <img src="/files/ftp_upload/2812/2812fig3.jpg" alt="図3"/> 図3。光ペプチドの分析物のピークプロファイル（赤）と重い安定同位体標識内部標準（青）を示す例のクロマトグラム。彼らは、クロマトグラフィーシステムから溶出としてY5光ペプチド（476.3&gt; 579.3）からの移行と標準（481.3&gt; 589.3）というラベルの付いた重い安定同位体のクロマトグラムは、時間の経過とともにプロットされます。

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利害の衝突は宣言されません。

として記述されているプロトコルの中で最も重要なステップは、ビーズが潜伏期間中によく混合のまま確保しています。ビーズがうまく/バイアルの底に沈降を許可すると、増加変動が発生します。それは潜伏期間の後にも/バイアルの上に残っている可能性のある液体を、スピンダウンすることも重要です。再現性のトリプシン消化も重要です。消化ここで説明する手順は、SISCAPAと一緒に私たちの研究室で広く利用されているが、それはおそらく消化の代替方法は、標的タンパク質の特定のセットに対して最適化することができますされ遊離抗体の15溶出がの検出を妨害しない。抗体は後でペプチドよりも列または溶出から除外されますが、抗体が前に大規模な実験でまたは遊離の抗体であれば親和性の濃縮にプロテインGビーズに架橋することができますので、可能性ペプチドは、問題となる。我々はまた、任意の残留ビーズまたは粒子を除去するために流れの逆方向（ローディングに比べて）でトラップカラムを洗浄するだけでなく、オートサンプラーでサンプルプレート下の磁石を配置することが有用な発見した。に記載の磁気ビーズのアプローチに加えて、技術はまた、列の形式（すなわちアフィニティークロマトグラフィー）に適合させることができます。12、16 ユーザーは、全体的なプロトコルと快適になると、技術はまた、全体的な分析を強化するいくつかの修正に適している。最初の11は 、それが濃縮ステップ（すなわち多重化）の抗体を組み合わせることにより、単一のアッセイで多検体を分析することが可能です。質量分析計は同時に分析対象物質の大量の分析が可能です。第二に、元のサンプルの音量を大きくすると、アッセイの感度を向上させます。

<table border="1"><tbody><tr><th>材料の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td> 1000ulディープウェル96ウェルプレート</td><td>エッペンドルフ</td><td> 951032786 </td></tr><tr><td>オアシスHLB 1ccのカートリッジプレート、をSep - Pak、40 mgを96ウェルプレート</td><td>ウォーターズ</td><td> 186003966 </td></tr><tr><td>キングフィッシャー96ウェルプレート</td><td>サーモフィッシャーサイエンティフィック</td><td> 97002540 </td></tr><tr><td> 96ウェル、白い壁のプレートをクリア</td><td> Bio - Rad社</td><td> HSP9601 </td></tr><tr><td> 96ウェルプレート用のカバーを阻止</td><td> Excelscientific </td><td> 12から169 </td></tr><tr><td> 96ウェルプレート用Axymatシーリングマット</td><td> Axygen </td><td> 521-01-151 </td></tr><tr><td> Xピアスフィルム</td><td>シグマアルドリッチ</td><td> Z722502 </td></tr><tr><td> PCRマグネットヘッド付カワセミ磁気ビーズプロセッサ</td><td>サーモフィッシャーサイエンティフィック</td><td> HSP 9601 </td></tr></tbody></table>表1：材料<table border="1"><tbody><tr><th>材料の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>尿素</td><td>シグマアルドリッチ</td><td> U6031 </td><td></td></tr><tr><td> Trizma基地</td><td>シグマアルドリッチ</td><td> T1503 </td><td></td></tr><tr><td>ジチオスレイトール（DTT） </td><td>ピアース</td><td> 20291 </td><td> &quot;スレイトール無重量&quot; </td></tr><tr><td>ヨードアセトアミド（IAM） </td><td>シグマアルドリッチ</td><td> I1149 </td><td></td></tr><tr><td>トリプシンゴールド</td><td>プロメガ</td><td> V5280 </td><td></td></tr><tr><td>プロテインG磁気ビーズ、2.8um </td><td>インビトロジェン</td><td> 10004D </td><td></td></tr><tr><td>リン酸緩衝生理食塩水（PBS） </td><td>サーモフィッシャーサイエンティフィック</td><td> L5401 </td><td></td></tr><tr><td> CHAPS </td><td>サーモフィッシャーサイエンティフィック</td><td> 28300 </td><td></td></tr><tr><td>ギ酸</td><td> EMD </td><td> 11670 </td><td></td></tr><tr><td>アセトニトリル</td><td>サーモフィッシャーサイエンティフィック</td><td> A998 - 1 </td><td></td></tr><tr><td>酢酸</td><td>シグマアルドリッチ</td><td> 242853 </td><td></td></tr></tbody></table>表2：試薬<table border="1"><tbody><tr><th>ソリューション</th><th>コメント（省略可能） </th></tr><tr><td> 100mMトリスpHを8 </td><td></td></tr><tr><td> 9 M尿素/ 100mMトリス（pH8）中で30mMのDTT </td><td>毎回新鮮に準備する必要があります</td></tr><tr><td> 100mMトリス（pH8）に500mMのIAM </td><td>毎回新鮮に準備する必要があります</td></tr><tr><td> 100mMトリスpHを8にトリプシン1mg/mL </td><td></td></tr><tr><td> 100mMトリスpHを8にトリプシンインヒビター1mg/mL </td><td></td></tr><tr><td>安定同位体標準マスターミックス</td><td></td></tr><tr><td>抗ペプチド抗体のマスターミックス</td><td></td></tr></tbody></table>表3：ソリューション用意する

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

タンパク質の測定で定量的なアッセイのための大きい必要性がある。主に定量のゴールドスタンダードと考えられ、伝統的なサンドイッチイムノアッセイは、、高コスト、長いリードタイム、および欠点（例えば異好性抗体、自己抗体干渉、&quot;フック効果&quot;）をはらんでいるに関連付けられています。1の代替テクニックは、親和性の濃縮です。定量的質量分析法と相まってペプチドから、一般的にSISCAPA（抗ペプチド抗体による安定同位体の標準とキャプチャ）と呼ばれる。このテクニック2、複数の/選択反応モニタリング質量分析法による安定同位体希釈や検出を有するペプチドの親和性の濃縮は、（ SRM / MRM - MS）は 、それぞれのタンパク質のための代用物としてのペプチドの定量的な測定を提供します。 SRM / MRM - MSがよく低分子化合物3、4の正確な定量のために確立されており、最近では血漿と細胞ライセート中のタンパク質の濃度を測定するために適応されています。タンパク質の定量を達成するために5-7、これらのより大きな分子はに消化されていますトリプシンなどの酵素を使用して、コンポーネントのペプチド。その配列はその種の標的タンパク質（すなわち&quot;proteotypic&quot;ペプチド）に固有の一つ以上の選択されたペプチドは、抗ペプチド抗体を用いて試料から濃縮およびサンプル中のタンパク質濃度の定量的な化学量論の代理として測定されます。したがって、安定同位体希釈（SID）の方法（すなわちスパイクインのペプチド標準ラベル安定同位体）に結合、SRM / MRMは、複雑な生体マトリックス中のタンパク質の定量のための代理としてproteotypicペプチドの濃度を測定するために使用することができます。アッセイは、従来のイムノアッセイに比べていくつかの利点があります。試薬が生成するために比較的安価な、分析物に対する特異性が優れている、アッセイは、高度に多重化、濃縮は、きちんとした血漿（ない枯渇が不要）から実行、および技術は、タンパク質や改造の広い配列に従順であることができることができます。興味深い。8-13このビデオでは、磁気ビーズのプラットフォームに適応としての基本的なプロトコルを示しています。

Watch this Scientific Journal Video about Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry at JoVE.com

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

安定同位体の標準と抗ペプチド抗体（SISCAPA）によって、それぞれのタンパク質の代用物としてのペプチドの定量的な測定を提供するために、安定同位体希釈質量分析法（MRM - MS）を有するペプチドの結合親和性の濃縮をキャプチャします。ここでは、部分的に自動化された形式で磁性粒子を使用してプロトコルを記述する。

質量分析法とあいまってペプチドイムノエンリッチメントを使用したタンパク質の定量化

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

quantification-proteins-using-peptide-immunoaffinity-enrichment

Research

JoVE Journal

Biology

22.7K ビュー.  Fred Hutchinson Cancer Research Center - FHCRC. 安定同位体の標準と抗ペプチド抗体（SISCAPA）によって、それぞれのタンパク質の代用物としてのペプチドの定量的な測定を提供するために、安定同位体希釈質量分析法（MRM - MS）を有するペプチドの結合親和性の濃縮をキャプチャします。ここでは、部分的に自動化された形式で磁性粒子を使用してプロトコルを記述する。

ビデオ: Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with 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 ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

スペクトル的に解決された二光子顕微鏡を用いたGタンパク質共役受容体相互作用の in vivo定量で

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

メチルトランスフェラーゼのプロファイリングおよびその他の S -アデノシル- L</subキャプチャの複合質量分析法（CCMS）別> -ホモシステイン結合タンパク質

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

同位体タグ質量分析法を用いたチオールレドックスプロテオームのプロファイリング

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

のタンパク質複合体の同定<em&gt;大腸菌</em&gt;タンデム質量分析との組み合わせでシーケンシャルペプチドアフィニティ精製を用いた

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕獲化合物質量分析 - 小説のc-ジ-GMPエフェクタータンパク質を同定するための強力なツール

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

絶対タンパク質定量のための質量分析法を選択反応モニタリング

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

質量分析のためのペプチドに組織および消化から細胞外マトリックスタンパク質の濃縮

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...