我々は、貴重なアドバイス、ディスカッション、および技術サポートのためにアランテッシェに感謝しています。私たちは、優れたサービスのためのコンコルディア大学の質量分析の生物学的応用のためのセンターを認めます。この作品は、CIHRとカナダのNSERCからの補助金によって支えられている。 VITはCIHR新調査官とゲノミクスのコンコルディア大学のリサーチチェア、細胞生物学と高齢化です。

材料および方法<ol><li>酵母菌株及び成長条件 野生型株BY4742（MATαhis3Δ1leu2Δ0lys2Δ0ura3Δ0が ）豊富なYEPD培地（1％酵母エキス、2％ペプトン、2％グルコース）で増殖させた。細胞を30℃で回転が5:1の&quot;フラスコの体積/培地体積&quot;比での三角フラスコで200 rpmで振盪しながら培養した。 </li><li>脂質の抽出のための酵母細胞の貯蔵 <ol><li>細胞の50 mlの培養液で5分間3000 × gで遠心分離によって回収した。 </li><li>冷たい水で2回洗浄した。 <ol style=" list-style-type:lower-alpha;"><li> 25mlの氷冷水でペレットを再懸濁します</li><li> 5分間3000 × gで遠心分離で細胞をペレット化</li></ol></li><li>ペレットを再懸濁し、エッペンドルフチュー​​ブに移す</li><li>遠心分離によってペレット、2分間に16,000 xgでは、使用するまで-80℃で上清と凍結を解除する。 （我々はイソプロピルアルコールのビーカーを使用するには、液体窒素を使用することもできます-80冷凍庫に保管） </li></ol></li><li>試薬 バイオテクノロジーグレードのクロロホルムとメタノールは、Sigma - Aldrich社から購入した。遊離脂肪酸とトリアシルグリセロールはLarodan（マルメ、スウェーデン）から購入した。リン脂質の様々な種 - ホスファチジルコリン、ホスファチジルエタノールアミン、ホスファチジルイノシトール、ホスファチジルセリン、ホスファチジン酸、およびカルジオリピンを含む - はアヴァンティ極性脂質（アラバスター、AL、米国）から入手した。テフロンライニングキャップを持つ高速ガラス遠心管はフィッシャーからのものであった。 </li></ol>脂質の抽出これは、ブライとダイアー8で記述されたプロトコルの変更です。抽出された脂質を持つすべての操作は、ガラスピペットやシリンジを用いて実施すべきである;彼らはクロロホルムと接触する場合、プラスチックには、大きなバックグラウンド信号を作成します。正確な量は、クロロホルムのため1:2:0.8の比率限り重要ではない-メタノール-クロロホルムのためのH 2、ステップ3でOと2:2:1.8 -メタノール-ステップ7でH 2 Oが保存されている。 <ol><li>氷冷蒸留H 2 Oを1.6 mlの凍結細胞を再懸濁し</li><li>高速ガラス遠心管に細胞懸濁液1.6 mlを。 </li><li>クロロホルムおよびMeOH（1:2）混合し、細胞懸濁液にガラスビーズを0.8mlの6mlを追加します。 </li><li>渦ガラスビーズを用いた細胞懸濁液に1分間2回。 </li><li>クロロホルム2mlを加え、穏やかに混ぜる。 </li><li>時々混合しながら室温で5分間インキュベートします。 </li><li>蒸留H 2 O 2 mlを加え、穏やかに混合する。 </li><li>時々混合しながら室温で5分間インキュベートする。 </li><li>室温で3000 xgで5分間遠心する。 </li><li>新しい高速ガラス遠心管に全体の液相を収集する。 </li><li>手順9からの細胞ペレットにクロロホルム3.2 mlを加え。 </li><li> 1分間ボルテックスを2回。 </li><li>室温で3000 xgで5分間遠心する。 </li><li>ステップ10から液相に上清を追加。 </li><li>室温で3000 xgで5分間遠心する。 </li><li>上段（水）相を破棄し、新しい高速ガラス遠心管に低い（有機）相を転送する。 </li><li>室温で3000 xgで5分間遠心する。 </li><li>ガラスバイアルに全体の上清を移し、窒素下で乾燥させます。 </li><li> -20℃で500のクロロホルム溶液とストア内脂質フィルムを溶かす</li></ol>質量分析による脂質の分析 0.1％のクロロホルム（1:1）（v / v）の水酸化アンモニウム - クロロホルム中の脂質の基準の株式のミックスは、表1と同様にメタノールの原液ごとに事前に準備する必要があります。 <ol><li>注入前に、表1に提供されて脂質の標準的なミックスを10μlとサンプル10μlを組み合わせる。午前1時01クロロホルムの200μLに：0.1％NH 4 OHとメタノール。 <ul><li>サンプルの比率への標準は、必要に応じて変更することができます。 </li></ul></li><li> 1μL/分の流速でナノエレクトロスプレーソースOSを搭載したマイクロマスQ - TOF 2質量分析計を用いて脂質を解決します。 <ul><li>正確なインストゥルメントのパラメータは、楽器から楽器ごとに異なります。ナノエレクトロスプレー源を装備したMicromass社Q - TOF 2（ウォーターズ、ミルフォード、MA、米国）のための設定については表2を参照してください。我々は質量分析計のQ - TOF型の高解像度の利点を取られているが、これは必須要件ではありません。 </li></ul></li><li>買収後はマススペクトルは平滑化され、背景が減算され、中央にして、ピークリストをExcelにエクスポート。各脂質クラスのピークは、それらの内部標準に正規化されます。アプリケーションによっては、そのようなdeisotopingとデコンボリューションなど、さらなる後処理を行う行う必要がある場合があります。 </li></ol> 表1内部の脂質基準、それらの濃度で標準的な混合、およびそれらの分析のためのMSモード。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>脂質クラス</td><td>標準チェーンの構成</td><td>標準の質量</td><td>濃度（μg/ ​​ml） </td><td> MSモード</td></tr><tr><td>ホスファチジン酸</td><td>午後2時/午後二時</td><td> 591.40 </td><td> 100 </td><td>マイナス</td></tr><tr><td>ホスファチジルエタノールアミン</td><td>午後2時/午後二時</td><td> 634.45 </td><td> 200 </td><td>マイナス</td></tr><tr><td>ホスファチジルイノシトール</td><td> N / </td><td> N / </td><td> N / </td><td>マイナス</td></tr><tr><td>ホスファチジルセリン</td><td>午後2時/午後二時</td><td> 622.37 </td><td> 40 </td><td>マイナス</td></tr><tr><td>カルジオリピン</td><td> 4x14：0 </td><td> 619.92 </td><td> 100 </td><td>マイナス</td></tr><tr><td>遊離脂肪酸</td><td>午前19時00分</td><td> 297.28 </td><td> 100 </td><td>マイナス</td></tr><tr><td>ホスファチジルコリン</td><td>午後2時/午後二時</td><td> 650.48 </td><td> 100 </td><td>正</td></tr><tr><td>トリアシルグリセロール</td><td>午前13時00分/ 13：00 /午後1時00分</td><td> 698.63 </td><td> 200 </td><td>正</td></tr></tbody></table>ナノエレクトロスプレー源を装備したMicromass社Q - TOF 2（ウォーターズ、ミルフォード、MA、米国） 表2。機器設定。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td>流量率</td><td>コー​​ン電圧</td><td>キャピラリー電圧</td><td>衝突ガス</td></tr><tr><td>ポジティブモード</td><td> 1μl/min </td><td> -28 V </td><td> 3.0 KV </td><td> 10 </td></tr><tr><td>負のモード</td><td> 1μl/min </td><td> 30 V </td><td> -3.2 KV </td><td> 10 </td></tr></tbody></table>

<ol>
	<li>Cao, H., Gerhold, K., Mayers, J. R., Wiest, M. M., Watkins, S. M., Hotamisligil, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+lipokine,+a+lipid+hormone+linking+adipose+tissue+to+systemic+metabolism.">Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.</a> Cell. 134, 933-944 (2008).</li><li>Claypool, S. M., Oktay, Y., Boontheung, P., Loo, J. A., Koehler, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+defines+the+interactome+of+the+major+ADP/ATP+carrier+protein+of+the+mitochondrial+inner+membrane.">Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane.</a> J. Cell Biol. 182, 937-950 (2008).</li><li>Guo, T., Gregg, C., Boukh-Viner, T., Kyryakov, P., Goldberg, A., Bourque, S., Banu, F., Haile, S., Milijevic, S., San, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+signal+from+inside+the+peroxisome+initiates+its+division+by+promoting+the+remodeling+of+the+peroxisomal+membrane.">A signal from inside the peroxisome initiates its division by promoting the remodeling of the peroxisomal membrane.</a> J. Cell Biol. 177, 289-303 (2007).</li><li>Russell, S. J., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+regulation+of+ageing.">Endocrine regulation of ageing.</a> Nat. Rev. Mol. Cell Biol. 8, 681-691 (2007).</li><li>Czabany, T., Athenstaedt, K., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+storage+and+degradation+of+neutral+lipids+in+yeast.">Synthesis, storage and degradation of neutral lipids in yeast.</a> Biochim. Biophys. Acta. 1771, 299-309 (2007).</li><li>Br&amp;uuml, g. g. e. r., Erben, B., Sandhoff, G., Wieland, R., &amp;amp, F. T., Lehmann, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+biological+membrane+lipids+at+the+low+picomole+level+by+nano-electrospray+ionization+tandem+mass+spectroscopy.">Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectroscopy.</a> Proc. Natl. Acad. Sci. USA. 94, 2339-2344 (1997).</li><li>Schneiter, R., Br&#252;gger, B., Sandhoff, R., Zellnig, G., Leber, A., Lampl, M., Athenstaedt, K., Hrastnik, C., Eder, S., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+ionization+tandem+mass+spectrometry+(ESI-MS/MS)+analysis+of+the+lipid+molecular+species+composition+of+yeast+subcellular+membranes+reveals+acyl+chain-based+sorting/remodeling+of+distinct+molecular+species+en+route+to+the+plasma+membrane.">Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.</a> J. Cell Biol. 146, 741-754 (1999).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can. J. Biochem. Physiol. 37, 911-917 (1959).</li></ol>

このメソッドは、容易に入手できる、安価な材料を使用して酵母のlipidomeの迅速な定量的評価が可能になります。これらの脂質は、水酸化リチウムと水酸化アンモニウムを置き換えることによって識別することができるが方法は、ジアシルグリセロール、ergosterolsとergosterylエステルを除いて、酵母細胞にある脂質の種のほとんどの同定が可能になります。説明する方法は、大きさの2〜3桁（脂...

a quantitative assessment of the yeast lipidome using electrospray ionization mass spectrometry

脂質は、生体分子の主要なクラスの一つであり、重要な役割の膜の力学、エネルギー貯蔵、およびシグナリングを再生<sup&gt; 1月4日</sup&gt;。出芽酵母<em&gt;サッカロマイセスセレビシエ</em&gt;、注釈付きのゲノムと非常にシンプルなlipidomeと遺伝的および生化学的に操作可能な単細胞真核生物は、多細胞真核生物の様々な脂質の種の生物学的機能を研究するための貴重なモデルです。<sup&gt; 2,3,5</sup&gt;。<em&gt; S.出芽酵母</em&gt;主に16または18個の炭素原子と不飽和のゼロまたは1度の鎖長を持つ脂質の10の主要なクラスがあります<sup&gt; 6,7</sup&gt;。脂質の同定と定量のための既存の方法 - そのような高速液体クロマトグラフィー、薄層クロマトグラフィー、蛍光顕微鏡、およびMSに続いてガスクロマトグラフィーとしては - 十分に確立が低感度、脂質の不十分別の様々な分子形式は、前の脂質誘導体化を必要と持っています分析に、または非常に時間がかかる可能性があります。ここでは、酵母細胞内の脂質の全体補数の同定および定量化のために調査スキャンESI / MSを使用して、これらの固有の限界を解決するために我々の実験手法の詳細な説明を提示する。記載されている方法は、それによって大幅にデータ収集のプロセスを加速し、酵母細胞から回収した複合脂質の混合物のクロマトグラフ分離を必要としません。このメソッドはg / mlとして低濃度で脂質の同定と定量を可能にし、成功した全酵母細胞およびそれらの精製された細胞小器官のlipidomesを評価するために適用されています。脂質の分析のこの方法を使用するための全体の酵母細胞からの脂質の抽出は、2〜3時間かかります。それは、ナノエレクトロスプレー源を装備したQ - TOF質量分析計で抽出、乾燥脂質の各サンプルを実行するためにわずか5〜10分かかります。

Watch this Scientific Journal Video about A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry at JoVE.com

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

我々は調査 - スキャンエレクトロスプレーイオン化質量分析（ESI / MS）を用いて酵母で数々の脂質の種を識別するための新しい定量的な脂質メタボロームの手法を説明します。このメソッドは、現在、脂質の同定と定量脂質の様々な分子形態を解決する能力で、感度、およびスピードで利用できるメソッドを超えています。

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 ...

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Research

JoVE Journal

Biology

11.6K ビュー.  Concordia University. 我々は調査 - スキャンエレクトロスプレーイオン化質量分析（ESI / MS）を用いて酵母で数々の脂質の種を識別するための新しい定量的な脂質メタボロームの手法を説明します。このメソッドは、現在、脂質の同定と定量脂質の様々な分子形態を解決する能力で、感度、およびスピードで利用できるメソッドを超えています。

ビデオ: A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization 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

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

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 ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

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 ...

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 ...

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 ...

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

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

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

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

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 ...

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 ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

出芽酵母におけるエンドサイトーシスの定量的、動力学的およびハイスループット分析のためのpHluorinの応用

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

シングル出芽酵母細胞での寿命と細胞表現型を測定するためのマイクロ流体デバイスを使用しました

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...