著者らは、米国エネルギー省（DEFG02 - 01ER15129）で、グラント番号0719232の下に米国国立科学財団によるこの作品の財政支援に感謝しています、とプロテアBiosciences社（ウェストバージニア州モーガンタウン）で。また、作者はプロトコルのビデオ撮影中に彼女の助けのためにジェシカA. Stoleeに感謝したいと思います。

次のプロトコルは、レーザーアブレーションのエレクトロスプレーイオン化（LAESI）実験の主なステップを説明し、動物や植物の組織サンプルの横方向と3次元（3D）イメージングのための代表的な例を示します。追加の実験的および技術的な詳細は他の場所から入手することができます。1-6 1。組織の準備と取り付け<ol><li>切片が必要な場合、組織は-20に-10℃10〜100μmの厚さのスライスにセクションにcryomicrotomeを使用° C以外の場合、特定の組織タイプに対して推奨されない限り。 </li><li>直接化学修飾のない平らな面（例えば、化学的に事前に洗浄したガラススライド）上のセクションをマウントします。分析中にすべての回で凍結組織を維持するために融解マウント時に区分された組織の場合は、セクションを融解マウントし、直ちにペルチェ冷却ステージに試料ホルダーを固定します。このステップは、セクション内の分子移動を最小限に抑える/防止するために必要とされる。 </li><li>必要に応じて、凍結した組織を維持するためにペルチェステージからの熱除去を容易にする低消費電力ファンを搭載したヒートシンクを使用してください。 </li><li>長時間（1-2時間）にわたって湿度の高い環境では、組織の表面に水または氷の凝縮のために検査する。組織上の水の結露が不利益LAESI実験の結像性能に影響を与えます。1 </li><li>必要に応じて、部屋の除湿機を使用したり、結露を防ぐために不活性ガス（例えば、乾燥窒素ガス）で満たされた環境チャンバー内に冷却サンプルを配置1。 </li></ol> 2。 LAESIイオン源の最適化 LAESIのイオン源は、中赤外レーザー、光ステアリング用光学素子のシリーズと焦点だけでなく、追加のサンプルホルダー、冷却コンポーネント、翻訳の段階、および溶媒送液システムで構成されています。図1は、質量分析計の大気圧イオン源の入り口に関してこれらの要素の典型的な配置を示しています。 <ol><li>図1は、位置、質量分析計のサンプリングコーン（d または- FP）のオリフィス以下のサンプル15〜20ミリメートルに示すように。 </li><li> 2.94μmの波長と10Hzの繰り返し率で中赤外レーザーを操作してください。 〜100μJ/パルスエネルギーのレーザーの出力を減衰させる。 </li><li>レーザーのパルスエネルギーは、試料表面に対して右の入射角を参照してください（垂直入射で試料にカップルに金色の鏡とレーザーの波長（例えば、平凸レンズCaF 2をまたはZnSeのレンズ）で透明な集光レンズの組み合わせを使用して、図1の）。 </li><li>質量分析計のサンプリングコーンのオリフィスの前で中間赤外線ビーム軸5〜8ミリメートルを置きます。 </li><li>フォーカスレンズの位置と焦点スポットに組織の除去を達成するためにレーザービームのパルスエネルギーを調整します。アブレーションボリュームの大きさは、画像処理アプリケーションのためにピクセル（または三次元画像のボクセル）のサイズを決定します。 </li><li>質量分析計の入口軸に沿って、と〜10 mmのオリフィスからエミッタ先端の距離（図1を参照）で、ナノスプレーエミッタを配置します。 </li><li>エレクトロスプレーの場合は、それぞれ、0.1％酢酸または正または負イオンモードのための0.1％酢酸アンモニウムの添加で50％メタノール溶液を調製。他の有機溶媒はアセトニトリル、イソプロパノール、などとして、、サンプルに応じて、分析タスクのための適切な濃度でメタノールを置き換えることができます。エレクトロスプレーの安定性は、成功するイメージングのための非常に重要です。溶媒の選択に応じて、流量、スプレー電圧は安定した噴霧を実現するために調整する必要があります。 </li><li>画像処理アプリケーションの反応性LAESI 6の場合は、electrosprayedソリューションは、反応物質が含まれている場合があります。 </li><li> 〜300 NL / minの流量でエレクトロスプレーエミッタを介してエレクトロソリューションを提供するためにシリンジポンプを使用してください。 </li><li>質量分析計のオリフィスが低電圧（&lt;500 Vグラウンドに対して測定）で保管されている場合は、エレクトロスプレーエミッタ（例えば、3,000 V）に直接高電圧を印加することにより、または金属労働組合を通じて、エレクトロスプレーを生成する。そうでない場合は、エレクトロスプレーを確立するために直接エレクトロスプレーエミッタまたは金属の組合を介してグランド。 </li><li> LAESIで最も効率的イオン生成のためにモードを噴霧円錐ジェットでエレクトロスプレーソースを操作する。噴霧モードとマススペクトルへの影響に関する操作変数の影響については、他の議論を参照してください。5,7,8 </li><li>慎重に同一平面上にレーザービーム、エミッタ、及びオリフィスの軸を保ちながらLAESIイオンの収量を最適化するLAESIのセットアップの相対距離を調整する。他の詳細な手順を見つけてください5。 </li><li>光学顕微鏡で、試料上のアブレーションのクレーターの横方向の寸法を決定する。 </li><li>三次元LAESIイメージング実験のために、個々のパルスでアブレーションを行い、例えば、使用してボクセルの深さを決定し、光学顕微鏡のZスタックモード3。 </li></ol> 3。分子イメージングとデータ解析イメージング実験では、組織試料は、より大きいまたはアブレーションのスポットの大きさに等しいステップサイズのXとY方向のレーザーの焦点面に移動されます。空間分解能は、入射レーザビームの集束によって制限されます。 <ol><li>試料表面上に関心領域を選択して、（X、Y）は、対応する境界の座標を取得します。 </li><li>グリッド化アルゴリズム（例えば、アダプティブグリッド、選択した領域のイメージング、長方形のグリッド、スパイラルパターン、Zスキャン、など）を選択撮像すべき領域の上に、各ピクセルでの滞留時間で試料表面をラスタになると選びました。 </li><li>三軸移動ステージ、所定のグリッドに従ってサンプルをラスタが可能なソフトウェアを使用してください。 </li><li>イメージングに必要な総時間を計算します。 </li><li>質量分析計のデータ取得の時間制限を無効にします。これが不可能な場合、計算された撮影時間の値にデータ収集の時間制限を設定します。 </li><li> LAESI横方向のイメージング実験を行うために、各ピクセルでの滞留時間内にマススペクトルで十分な信号対雑音比を生成するために適切な繰返しレートでの中赤外レーザ光源を開始します。 3D分子イメージングのために、成功し、単一のレーザーパルス内で生成されたイオンを大量に分析するためにレーザー光源の繰り返しレートより高いスペクトルの取得率を使用してください。アブレーションのシーケンスを開始するSTART信号を待ちます。 </li><li>エレクトロスプレー源をオンにします。イメージングに必要なフルタイムのための十分な解決策があることを確認してください。 </li><li>同時にマススペクトルの取得、中赤外レーザーアブレーション、および表面のスキャンを開始します。 </li><li>イメージングの実行が終了すると、表面のスキャン、中赤外レーザー発振、及びデータ収集を停止します。 </li><li>レーザ光源を無効にします。 </li><li>高電圧の電源を切ります。 </li><li>シリンジポンプの電源を切ります。 </li><li> STANDBYモードに質量分析計を設定します。 </li><li>ペルチェ冷却用電子機器の電源を切ります。 </li><li>使用する場合は不活性ガスの流れを閉じます。 </li><li>横方向のイメージングまたは対応するスペクトルを持つ3次元解析におけるボクセル内のピクセルの絶対座標を相関させるソフトウェアを使用してください。 </li><li>横方向と3D分子画像を得るために分析の絶対座標に対して選択されたm / z値のためのイオン強度の信号をプロットします。 </li></ol> 4。代表的な結果図2は、いくつかの主要な組織型と画像診断法の代表的な結果が得られます。パネルには、脱水症状を防ぐために、実験中に凍結されている動物の組織切片のためのケースを示しています。1さらには 、サンプルを試料表面に結露から周囲の水の蒸気を避けるために乾燥窒素ガスの環境に位置していた。ラットの脳の100μm厚の冠状セクション（ ドブネズミ ） が横方向にLAESIで撮像した。および/ ​​またはPE（O - 36：3） のm / z 728.559と：脳の解剖学的領域は、（パネルの光学画像を参照）plasmalogensのPC（3 O - 33）について得られた分子像と良い相関を示しています。 パネルBは、Zebra工場（ アフェランドラsquarrosa）葉組織の3次元LAESIイメージングを示しています。葉が脱水症状に対する自然の防御機構を持っているため、サンプルは、周囲の環境で調べることができる3。得られた3次元分子画像は、プライマリおよび二次代謝産物の配布さまざまなパターンを明らかにした。他の人の中で、 はm / z 285.076とacacetinは、他の均一な分布と上から2番目と3番目の層の黄色の分野で高いイオンカウントで検出された。この分布は、光学像に見られる斑入りのパターンと一致した。 <img src="/files/ftp_upload/2097/2097fig1.jpg" alt="図1" /> 図1。 LAESIシステムの概略図 （ES、エレクトロスプレーエミッタ先端、または、質量分析計のサンプリングコーンのオリフィス、FL、レンズを中心に、FP、フォーカルポイント、P、ペルチェ冷却ステージ、HS、ヒートシンク）。分子と試料のイオン（緑色のドット）を接種した帯電液滴を生成するためにエレクトロと中赤外アブレーション（赤い点）合体中に排出される粒子状物質の部分。これらの液滴から放出イオンが質量分析計で分析して記録されます。 <img src="/files/ftp_upload/2097/2097fig2.jpg" alt="図2" /> 図2。横方向と3D想像力のための代表的な結果LAESI質量分析とNG（A）上部パネルは、ラットの脳の光学像（ ドブネズミ ） を描く冠状セクションとplasmalogens PC（O - 33：3）で得られた分子のイメージ。および/ ​​またはPE（O - 36 ：3） のm / z 728.559。白いスケールバーは1mmに相当します。からの許可（参考1）に適応。著作権2010 American Chemical Societyの。 （B）下のパネルには、多彩なゼブラプラント（ アフェランドラsquarrosa）から葉の3次元画像を示しています。 のm / z 285.076とAcacetinは、他の均一な分布と上から2番目と3番目の層の黄色の分野で高いイオンカウントで検出された。からの許可（参考文献3）を得て転載。著作権2009 American Chemical Societyの。

<ol>
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利害の衝突は宣言されません。

異なる組織型はこれらの影響を緩和するために順番に、試料のアブレーション特性に影響を与えることができる、含水率と引張強度、9はさまざまです、それは間を変更するときにレーザーのフルエンス、サンプルの処理、および分析のプロトコルが改訂されることが望まれている主要な組織の種類。 単一のセルまたはより高い解像度の調査のため、中赤外光の代わりに、フォーカスレンズのシャープ、光ファイバに結合することができます。10は組織内の選択したセルのすぐ近くに光ファイバの先端を配置することにより、LAESI分析はで行うことができます。単一細胞レベル。 ラベルフリー周囲イオン化質量分析用イオン源として、11 LAESIは組織中の生化学的プロセスの研究に大きな可能性を示している。直接解析、横方向と3次元イメージングの利点を得ることも、LAESIは、プロファイリングと同様に画像処理アプリケーションのための新興バイオ分析ツールです。

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<td>Mass spectrometer</td>
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<td>Waters Co. (Milford, MA)</td>
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<td>Mid-IR laser</td>
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atmospheric-pressure molecular imaging of biological tissues and biofilms by laesi mass spectrometry

質量分析法で、周囲のイオン化法は、分析調査がネイティブのような実験条件下では組織やバイオフィルム上で直接実行することができます。レーザーアブレーションのエレクトロスプレーイオン化（LAESIは）そのような開発であり、特に水を含む試料の調査に適しています。 LAESIはサンプルの水分子を励起する中赤外レーザービーム（2.94μmの波長）を利用しています。アブレーションのフルエンスのしきい​​値を超過した場合、サンプルの材料は、粒子状物質の形で追放し、これらの発射は、試料表面上のミリメートルの数十に移動される。 LAESIでは、このアブレーションプルームが放出された試料の一部をキャプチャし、気相イオンにその化学成分を変換するために高度に帯電した水滴により代行受信されます。大気圧イオン源のインタフェースを装備した質量分析計は、試料のプローブ領域（ピクセル）に由来する放出イオンの組成を分析し、記録するために採用されている。画素の配列に対する体系的な尋問は微小分析モードでの分子イメージングへの道を開きます。 LAESIの質量分析イメージングのユニークな点は、横方向のイメージングとの組み合わせで、3次元（3D）分子イメージングを可能にする、深さプロファイリングです。 〜100μmであり、それぞれ〜40μmの、の現在の横方向と深さの解像度で、LAESI質量分析イメージングは​​、生体組織の分子構造を探索するのに役立ちます。ここで、我々はLAESIシステムの主要な要素を確認し、成功するイメージングの実験のためのガイドラインを提供しています。

Watch this Scientific Journal Video about Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry at JoVE.com

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

レーザーアブレーションのエレクトロスプレーイオン化（LAESI）は質量分析用大気圧イオン源です。イメージングモードでは、中赤外レーザーのプローブは組織切片またはバイオフィルム全体の分子の分布。この手法は、ネイティブの実験条件の下で実施、多様な生物分析研究のための新しいアプローチを提示。

LAESI質量分析による生体組織とバイオフィルムの大気圧分子イメージング

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

atmospheric-pressure-molecular-imaging-biological-tissues-biofilms

Research

JoVE Journal

Biology

14.1K ビュー.  George Washington University. レーザーアブレーションのエレクトロスプレーイオン化（LAESI）は質量分析用大気圧イオン源です。イメージングモードでは、中赤外レーザーのプローブは組織切片またはバイオフィルム全体の分子の分布。この手法は、ネイティブの実験条件の下で実施、多様な生物分析研究のための新しいアプローチを提示。

ビデオ: Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI 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

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 ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T波イオンモビリティー質量分析：タンパク質複合体解析のための基本的な実験手順

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

大気圧質量分析法による単一細胞の直接分析

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

シングルプローブの応用：周囲条件下での質量分析イメージングと単一細胞解析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

そのセンターの蛍光質量イメージングによるゴルジ体タンパク質の量的なローカリゼーション

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高圧急速凍結、マイクロ波を用いたコントラスト強調、およびボリューム イメージングのために埋め込む最小限の樹脂による生体試料作製

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...