この研究は、サイモンフレーザー大学のスタートアップ資金によって支えられてきた。

1。収穫膜<ol><li>細胞ペレット（〜10 8細胞）にプロテアーゼ阻害剤カクテル低張緩衝液（10mMトリス-HCl、10mMのKCl、1.5mMのMgCl 2、pH8.0）に加え、氷上で15-30分間インキュベートする。溶解注射針（5月10日パス）に試料を通すか、ダウンスホモジナイザー（15〜30ストローク）することにより、試料を均質化することにより、細胞。溶解の効率性をチェックするために、血球計数器およびトリパンブルー染色を使用してください。 </li><li> 3000 GX 15分で溶解液の微分遠心分離によってミクロソーム画分を取得し、100,000 GX 2時間。最初の遠心分離は、核画分でペレットを取得し、上清のその後の遠心分離は、細胞膜ならびに膜結合オルガネラ8を含有するミクロソーム画分である第二のペレットを生成する。最終的な上清を細胞質画分である。さらにanalysために-80℃の冷凍庫にミクロソーム画分を保存以下に示すとおりである。 </li></ol> 2。、変性を溶かし、ダイジェスト膜タンパク質<ol><li>変性緩衝液（40mMトリス、10mMのEDTA、10mMのTCEP、0.5％Rapigest、pH8）中でミクロソーム画分を溶解し、次いで、10分間100℃で溶液をインキュベートする。 </li><li>室温に加熱し、溶液を冷却し、8 Mの最終濃度に溶液に超高純度尿素粉末を添加し、30分間37℃でサンプルをインキュベートする。 </li><li> 15mMの最終濃度まで試料にヨードアセトアミドストック溶液を追加し、サンプル中の遊離チオールをアルキル化するのに室温で30分間、暗所で溶液をインキュベートする。 10mMの最終濃度まで試料にDTTストック溶液を追加し、過剰のヨードアセトアミドをクエンチし、室温でさらに10分間溶液をインキュベートする。 </li><li> pH8の40mMトリス緩衝液で得られた溶液を10倍希釈し、続いてtrypsi 1:20の比率で試料にトリプシンを加える総蛋白のN。反応が完了したことを確認するために37℃のオーブンで一晩中消化反応を維持する。 </li><li> HClでpH1に試料溶液、また洗剤を分解条件、 すなわち Rapigestを酸性化することによって消化を終了します。 1時間37℃でRapigestを低下させ、遠心分離によって開発された降水量を削除します。 </li><li> C-18固相抽出（SPE）カートリッジにより、サンプルのペプチドが含まれており、スピードバックで得られた試​​料を乾燥上清を清掃してください。 </li><li>前および消化効率を確認するために、トリプシン消化後の試料のSDS-PAGE分析を実行する。 </li></ol> 3。糖ペプチドキャプチャ<ol><li>カップリングバッファー（100 mM酢酸ナトリウム、pH 5.5）中で洗浄さペプチドを溶解する。室温で、暗所で30分間10 mMの最終濃度でペプチド溶液に過ヨウ素酸ナトリウムを追加します。これは、アルデヒドにグリカンシス - ジオール基を酸化する、ヒドラジド化学を介して樹脂に結合するためにグリカンを可能にする。室温で10分間、20mMの最終濃度およびpH 5で亜硫酸ナトリウムによって過度の過ヨウ素酸を消光する。 </li><li>樹脂に結合糖ペプチド1:4の比（溶液への樹脂）でのペプチド溶液にヒドラジド誘導体化樹脂をご紹介します。完全なカップリングのためのエンドツーエンドの回転に1〜2日間、37℃で反応をインキュベートする。 </li><li>以下の各1mlで二回、樹​​脂を洗浄することにより未結合ペプチドを除去：DI水、1.5 MのNaCl、メタノールおよび80％アセトニトリル。最後に、100mMのNH 4 HCO 3へのシステムのバッファを交換するためにpH8の100mMのNH 4 HCO 3 1mlで3回、樹脂を洗浄する。 <ol><li> （オプション）未結合のペプチドの分析のための上清および洗浄を収集します。 </li></ol></li><li> 37℃で一晩のインキュベーションでのPNGアーゼFにより、樹脂からのN-グリコペプチドをリリースnはエンドツーエンドで回転。遠心分離し、80％のアセトニトリル洗浄によってリリースペプチドを収集します。 LC-MS分析のためのスピードバックで得られた溶液を乾燥させる。 </li></ol> 4。さらに分別（オプション） さらに、試料の複雑性を簡略化するために、得られたN-グリコペプチドを分画する。例えば10mmアンモニア蟻酸、20％アセトニトリルを用いてpHを3に乾燥したペプチドを再溶解し、ペプチドを分画する強力な陽イオン交換（SCX）クロマトグラフィーを使用しています。得られた溶出液を乾燥し、次いで逆相LC-MS 8,17,18により得られたペプチド画分を分析する。 放出されたN-グリコペプチドの5。クリーニング（オプション） 懸念は、ペプチドの汚染の可能性のために上昇した場合、0.1％ギ酸に乾燥したペプチドを再溶解し、さらに、逆相LC-MS分析の前にペプチドをきれいにするMCX SPEカラムを使用しています。 <stronG&gt;注：データベース検索のパラメータを。 樹脂オフのN-グリコペプチドの選択的切断時には、PNGアーゼFは、アスパラギン酸とN-グリカンリンクアスパラギンに変換します。そのため、遊離されたN-グリコペプチドの0.9840ダマスシフトがあります。正確にこれらのペプチドを同定するために、この変更は、メチオニンのシステインおよび酸化カルバミドメチル化などの一般的な改変を加えた検索パラメータに追加する必要があります。

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

ここでは、細胞表面タンパク質のプロファイリングのためのグリコペプチド捕獲戦略をご紹介します。この方法は、血液中、ならびに他の体液中または細胞培養培地中のもののような分泌タンパク質を研究するために適用することができる。 この方法の成功は、サンプルの完全消化に依存しています。従って、消化効率のSDS-PAGEの特徴付けは、特に、試料を初めて分析の...

細胞表面タンパク質は、細胞外環境と相互作用し、細胞の外側から内側への信号を中継する。従って、細胞外マトリックスタンパク質を含む、これらのタンパク質は、増殖、成長、移動、分化などから老化及び範囲細胞生物学及び生理学の全ての局面において重要な役割を果たす。表面タンパク質は、他の細胞、タンパク質および小分子3と相互作用することによって機能する。薬物の60％以上が細胞表面タンパク質4を標的とするような細胞表面タンパク質の分子特徴付けは、生物学者のためだけでなく、製薬会社のためだけでなく、非常に興味深い。 タンデム質量分析（MS）は、その優れた感度、精度、およびタンパク質およびペプチドの同定のためのスループットで、グローバルプロテオミクス研究5,6のための強力なツールとなっている。しかし、表面タンパク質として、MSに基づくプロテオミクスの重要な課題を提起ほとんどの表面タンパク質は、低量で重い変形が存在する。表面タンパク質の膜貫通領域は、それらを疎水性;これは、特にマルチパス膜貫通タンパク質の場合である。これは、界面活性剤の助けを借りずに、水溶液中の膜タンパク質を溶解することが困難である;しかし界面活性剤の使用は一般的にタンパク質の同定において、HPLCとMS 1,7,8の性能を抑制します。したがって、膜タンパク質が不十分直接LC-MSベースのプロテオミクスに特徴付けられている。 グリコシル化は、細胞表面タンパク質9で行われている最も重要かつ豊富な翻訳後修飾の一つである。グリカンの膨大な複雑さと不均一性は、ペプチドのMSシグナル10を妨げる。それにもかかわらず、いくつかのプロテオミクスの方法は、表面タンパク質を濃縮する前LC-MS分析にタンパク質から糖部分を除去するために、このユニークな変形例を使用している。これらの方法Sは、レクチンベースの親和性捕捉11とヒドラジド系、ホウ酸系化成キャプチャ12と同様に親水性クロマトグラフィー分離8,13が含まれています。グリカンの除去は、通常のタンパク質に膜タンパク質を変換し、劇的に、MSの特性評価を簡素化します。グリコシル化はまた、膜タンパク質とは対照的に、高い溶解性を有する分泌タンパク質で起こるので、多くのglycoproteomic方法は、可溶性タンパク質用に最適化され、膜タンパク質8,14に配備されたときに低い糖ペプチドの選択性および感度を有する傾向がある。他の方法も豊かにするために存在し、特に、超遠心分離15および標識戦略16を用いたものなどの細胞表面タンパク質。膜タンパク質を特徴付けるための手法や他の既存の方法の間の詳細な比較は最近17を実施し 、その結果は我々の手法は、同様に良好に実行できることが示され、もしされませんでしたすべて比べ膜プロテオミクス方法よりも、より良い、より高いシンプルさ。 研究者はこのメソッドを使用しやすくするため、ここでは詳細に一般的なプロトコル。この方法は、既存のグライコ戦略のいくつかの利点を統合し、膜糖タンパク質のために特別に考案されて、まだ方法は、分泌タンパク質についても同様に動作します。この方法の特徴は、1）膜タンパク質の完全な可溶化は、2）の代わりに固体撮像基板を用いた場合、潜在的な立体障害を排除する糖タンパク質の糖ペプチドの濃縮は、3）ヒドラジド化学の使用は、共有結合との間を形成すること結合したグリコペプチドは、高glycoselectivityためのストリンジェントの洗浄に耐えることができ、4）能力が減少したサンプルの損失短縮手順の持続時間のために一本のチューブにキャプチャ全体の手順を実施するように、糖ペプチドおよび撮像基板。変数を研究するためにこのメソッドを実装した後細胞及び組織を含む生物学的試料のETY、我々は、糖タンパク質8,17,18に対して高い選択性（&gt; 90％）を観察した。

<table border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DTT</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646563</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCEP</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodoacetamide</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A3221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rapigest SF</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186001860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium periodate</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">311448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNGase F</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:119px;">P0704S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Affi-Gel Hz Hydrazide gel</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:119px;">153-6047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:199px;">Worthington Biomedical</td>
			<td style="width:119px;">LS02115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sep-Pak C-18 cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">WAT054955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor coctail</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:199px;">Amersco</td>
			<td style="width:119px;">568</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium sulphite</td>
			<td style="width:199px;">Caledon</td>
			<td style="width:119px;">8360-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Invertase</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">I0408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alpha-1 trypsin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ribonulease B</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">R7884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Avidin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ovalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">C0755</td>
			<td></td>
		</tr>
	</tbody>
</table>

glycopeptide capture for cell surface proteomics

細胞外マトリックスタンパク質を含む細胞表面タンパク質は、例えば、増殖、分化、および増殖などの全ての主要な細胞プロセスおよび機能に関与する。これらのタンパク質の総合的特徴付けは、バイオマーカー探索、細胞型の識別、および薬剤ターゲットの選択だけでなく、細胞生物学や生理学の我々の理解を進めるために支援するための豊富な情報を提供します。表面タンパク質は、しかし、本質的に低いため、その存在量、高い疎水性、及び重翻訳後修飾のため、有意な分析的な課題を提起する。表面タンパク質に普及してグリコシル化を利用して、我々はここでいくつかの既存のN-グライコ手段の利点を統合し、高スループット糖ペプチド捕獲アプローチをご紹介します。本手法は、表面タンパク質由来の糖ペプチドを豊かにし、LC-MSを用いて容易なプロテオミクスのために彼らのグリカンを削除することができます。解決のN-glycoproteomeはINFと、タンパク質の同一性および量だけでなく、糖鎖付加の自分のサイトのormation。この方法は、癌、幹細胞、および薬物毒性を含む領域における一連の研究に適用されている。この方法の限界は、試料の比較的多量のサイトゾルタンパク質を中心に研究と比較してこの分析のために必要とされるように、表面膜タンパク質、低存在量である。

実験手順の代表的なフローチャートを図1に要約されている。標識およびさらなる分画工程は任意であり、詳細は最近の刊行物18に記載されている。別のオプションは、樹脂と反応しない非修飾ペプチドを分析することである。変更されていないペプチドを分析することの利点は、そのようなタイトジャンクショ​​ンにおけるクローディンのような非グリコシル化ペプチ...

Watch this Scientific Journal Video about Glycopeptide Capture for Cell Surface Proteomics at JoVE.com

Glycopeptide Capture for Cell Surface Proteomics

細胞表面タンパク質は、生物学的に重要かつ広範にグリコシル化されている。我々は、可溶化豊かに、そして容易なLC-MSベースのプロテオミクス解析のための脱グリコシル化は、これらのタンパク質には、ここグリコペプチド捕捉アプローチをご紹介します。

細胞表面プロテオミクスのための糖ペプチドキャプチャ

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

glycopeptide-capture-for-cell-surface-proteomics

Research

JoVE Journal

Biology

11.2K ビュー.  Simon Fraser University. 細胞表面タンパク質は、生物学的に重要かつ広範にグリコシル化されている。我々は、可溶化豊かに、そして容易なLC-MSベースのプロテオミクス解析のための脱グリコシル化は、これらのタンパク質には、ここグリコペプチド捕捉アプローチをご紹介します。 

ビデオ: Glycopeptide Capture for Cell Surface Proteomics

Cell Capture Using a Microfluidic Device

マイクロ流体デバイスを用いた細胞のキャプチャ

生物学

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

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 ...

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 ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

マンモスフィアの自然発生の事前侵襲乳房病変の非酵素的、無血清組織培養

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

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 ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

表面拡散と酵母染色体の免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

磁気ビーズを使用した機械的張力に応答して細胞表面接着リフォームの分析

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...