This work is supported by the Genomics Research Center, Academia Sinica and the Ministry of Science and Technology of Taiwan, the Republic of China (Contract No. 104-2119-M-001-014).

注意：このプロトコルは、乾燥液滴法を用いて調製した（1-7）マルトトリオース及びブラジキニンフラグメントの空間的な不均一性を減少させるために開発されています。プロトコルは、調製及び前処理、試料堆積、乾燥、及び質量分析データ分析を含む三つの主要な段階からなります。手順は、以下でより詳細に概説し、説明します。 1.準備と前処理<ol><li>サンプルプレートのクリーニング<ol><li>ニトリル手袋を着用し、洗剤と蒸留脱イオン水（DDW）で穏やかにサンプルプレートを手で洗います。 </li><li>メタノール（MeOH）でとDDWでサンプルプレートをすすぎます。 </li><li> 600ミリリットルのビーカーにサンプルプレートを挿入し、DDWでいっぱい。 </li><li>超音波浴（200 W、40キロヘルツ）で15分間、DDWでサンプルプレートを超音波処理します。 </li><li>ビーカーからDDWを削除し、MeOHでビーカーを埋めます。 </li><li>超音波浴（200 W、40キロヘルツ）で15分間、MeOH中でサンプルプレートを超音波処理します。 </li><li>窒素ガスをプレート上で溶剤滴を吹き飛ばすとサンプル堆積前にサンプルプレートの乾燥を保ちます。 </li></ol></li><li>乾燥チャンバーの温度を調節します 注：乾燥室は35×20×45 cm 3である（幅×奥行き×高さ）アクリルチャンバー図1は、この乾燥システムの写真を示します。チャンバは、乾燥室の内部に設置較正湿度計によって監視低い相対湿度を維持するために一定の流量でガス流量計を介して室温窒素ガスでパージされます。プログラムされた恒温水循環装置を備えた乾燥室中の銅ベースブロックは、ステンレス鋼サンプルプレートを収容するために使用されます。銅ベースブロックを25℃に5からのサンプルプレートの温度を調節することができます。空気、銅ベースのブロック、及びサンプルプレートの温度は、K型熱電対によって監視されます。 <ol><li>ドアを開け、急速に銅をサンプルプレートを置きますベースブロックは、その後、ドアを閉じます。 </li><li>手動で時間当たり10標準立方フィート（SCFH）で窒素流量を設定するためにガス流量計を調整します。 </li><li>相対湿度が常に25％未満である保証するために、湿度計と微調整ガス流量計による乾燥チャンバ内の相対湿度を監視します。 </li><li> K型熱電対によってサンプルプレートの温度を監視し、サンプルプレートは、実験又は対照を室温（25℃）が5℃になるまで手動で水循環温度を調整します。 注：設計温度でサンプルプレートを安定させるために、水循環装置の温度は、典型的には、設計されたサンプルよりも低い0〜5℃に設定されています。例えば、サンプルプレートに5℃を維持するように、水循環装置の温度設定は、0℃〜2の範囲です。 25℃でのサンプルプレートを維持するために、水循環装置の温度設定は23℃〜25℃の範囲です。 </li><li>必要な温度と相対湿度はサンプル堆積の前に（ 表1）に達していることを確認します。 注：異なるサンプルプレートの温度が乾燥プロセスのすべてのパラメータ、ならびにそれらの設定値を表1に示します。 注：室のドアを長時間開いている場合は、低いサンプルプレートの温度では、サンプルプレート上の結露が発生することがあります。結露が発生した場合は、ドアを閉じて、結露が出て乾燥するまでの任意のサンプルを堆積しないでください 。 </li></ol></li><li>マトリックスと分析物溶液の調製<ol><li>マトリックス溶液の調製<ol><li> 50％DDW水溶液：50％アセトニトリル（ACN）と0.1 M THAP溶液を調製します。 </li></ol></li><li>検体の準備<ol><li> DDWで10 -4 Mマルトトリオース溶液を調製します。 </li><li> 10 -5 Mのブラジキニン断片（1-7）溶液を50％アセトニトリル中で調製します（ACN）：50％DDW水溶液。 </li></ol></li></ol></li></ol> 2.サンプル沈着と乾燥<ol><li>プレミックス0.1 MのTHAP液の0.25μlの10 -4 Mのマルトトリオースまたは10 -5 Mブラジキニンフラグメント（1-7）マイクロ遠心チューブ内のソリューションの0.25μlの。 </li><li> 3秒間混合溶液をボルテックス。 </li><li>遠心管の底に溶液を回収するために2秒（2,000×gで）混合液を遠心分離。 </li><li>乾燥室のドアを開け、慎重にピペットでサンプルプレート上に溶液0.1μLを堆積し、すぐにドアを閉めます。 </li><li>試料液滴が乾燥するのを待ちます。 注：典型的に観察された乾燥時間の異なるサンプルプレートの温度を表1に記載されていると5℃のサンプルプレートの温度は、平均乾燥時間が800〜1000秒です。 25°Cのサンプルプレート温度について、平均乾燥時間は100〜150秒ですEC。 </li><li>乾燥させた後、乾燥室の扉を開きます。 </li><li>室温（25℃）での水循環温度を設定します。 注：サンプルプレートは、乾燥工程中に、室温（25℃）で常に保たれている場合は、このステップをスキップします。 </li><li>室温（25℃）にサンプルプレートの温度が戻った後、乾燥室からサンプルプレートを取り外します。 </li><li> 5Xの実体顕微鏡下でのサンプルの形態を調べ、スナップショット明視野像を取ります。 注：予想通りの結晶形態ではない場合は、同様の手順を使用して新しいサンプルを用意する必要があります。典型的な結晶形態は、図2の上のパネルに示されています。 注：このような5°Cなどの低サンプルプレート温度、との例では、乾燥室からそれを取る前に室温にサンプルプレートをウォームアップすることが重要です。サンプルを堆積すると、予混合solutioを保管しないでください10秒以上のピペットの先端中のn。サンプルを堆積した後、再び予混合ソリューションを使用しないでください 。 図2の上のパネルは、異なるサンプルプレートの温度を用いて調製した試料の明視野画像を示します。 </li></ol> 3.質量分析データ解析<ol><li>質量分析データ集録 注：調製後、試料をイメージング質量分析を用いて分析することができます。現在の研究では、イメージングMS実験を実験室内蔵同期デュアル極性TOFを使用して行われる（DP-TOF）イメージング質量分析計。撮像能力を持つ15市販MALDI-TOF質量分析計は、また、そのような実験に適しています。質量分析計は、線形抽出と最適化された抽出遅延を有する陽イオンモードで操作されます。イオンの運動エネルギーは、20 kVです。レーザービームの大きさは、試料表面上の直径が35μmであり、すべてのスポットのスペクトルAVEあります5レーザーショットの怒り。 <ol><li> MALDI質量分析計にサンプルプレートを挿入します。 </li><li>ステップ2.1から2.9に調製した試料を撮像する質量分析を実行します。 </li><li>結果ウィンドウに表示される質量リストから特有の質量ピークを選択して、2次元イオン画像をプロットする「2D」をクリックします。 注：THAPと混合マルトトリオースのために、特徴的なピークは、マルトトリオース、プロトン化THAPをナトリウムイオン付加し、THAPをナトリウムイオン付加されています。 THAPと混合ブラジキニン断片（1-7）について、特徴的なピークは、ブラジキニンフラグメント（1-7）、プロトン化THAPをプロトン化、およびTHAPをナトリウムイオン付加が含まれます。 </li><li>信号強度の上限と下限を決定するために、ポップアップウィンドウに調整ボタンをクリックして、「画像を保存」をクリックします。この設定は、イオンの画像のコントラストを定義します。 注：データのすべての個々のセットでは、ひび割れ領域及び低輝度を示すヌルスポットが除去されます。 </li><li>イオンを観察し、比較しますステップ2.9で撮影された明視野像と画像。 注記：特定のイオンの画像のイメージング質量分析法及び構造は、市販の機器を使用して達成することができます。データ収集および解析ソフトウェアの様々な、ユーザーは、高品質の画像を取得するために、機器ベンダーが提供するソフトウェア命令に従ってください。 </li></ol></li><li>データ分析 注：サンプルの不均一性を定量的に分析されます。このデモでは、すべてのサンプルは、イオンの空間分布を分析するために、社内で開発されたソフトウェアにより、複数の同心円状の領域に分割されています。分析はまた、スタンドアロンのデータ分析ソフトウェアを用いて行うことができます。 <ol><li>重要でない領域を除去するために、結果ウィンドウに表示されているイオン像にヌルスポットやひび割れ領域をクリックしてください。 注：この手順では、イオン像の本質的な領域を定義します。 </li><li>イオン像の最外層を見つけるために、「エッジを見つける」ボタンをクリックします。 </li><li>データベース内の最も外側の層のイオン存在情報を保存し、同時にイオン像から、この層を除去するために、「控除」をクリックします。この最外層を表すチェックボックスが結果ウィンドウの「出力データ」リストに表示されます。 </li><li>イオン像の中心が定義されるまで繰り返して、3.2.2と3.2.3を繰り返します。 </li><li>クリックして、「出力データ」リストのすべてのチェックボックスを選択して、データをエクスポートする「エクスポート」をクリックします。 </li><li>イオンの空間分布情報を取得するために、すべての層の平均イオン存在を計算するために、表計算ソフトを使用してエクスポートしたデータを開きます。 </li></ol></li></ol>

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The authors declare no competing financial interest.

以前の理論的予測に基づいて、液滴内の温度誘導性の流体力学的な流れは、溶媒の蒸発によって誘導される外向きの毛細管流れを克服することができます。温度は、液滴増大内勾配時の分子のような内部再循環の効率が向上します。周囲温度でその周囲を維持しながら、5℃下、サンプルプレートの温度を維持した場合に予測された結果によれば、液滴内の再循環流の平均速度は、外側にキャ...

Mass spectrometry (MS) is one of the most important analytical techniques for analyzing the molecular compositions of complex samples. Among all the ionization methods used in MS, matrix-assisted laser desorption/ionization (MALDI) is the most sensitive and widely used method in bioanalytical applications.1 In comparison to other ionization techniques, MALDI has the highest sensitivity and high tolerance to salt contaminants. Such analytical properties make MALDI the first choice for carbohydrate analysis and many proteomics applications. However, sample preparation is a crucial step for obtaining high quality data in MALDI-MS.
The most commonly used sample preparation method for MALDI-MS is the dried-droplet method, in which sample droplets are deposited on a surface and dried under ambient conditions. This drying method is simple and generally effective.2-5 However, a common problem in the dried-droplet method is that the resultant analyte/matrix crystals normally distribute irregularly. In many cases, the crystals aggregate at the periphery of sample areas, resulting in the so-called ring-stain formation.6-8 The heterogeneous crystal morphologies affect the spatial distribution of analyte molecules, which results in severe fluctuation in ion signal over sample areas. Such severe signal fluctuations and poor data reproducibility are known as the "sweet spot" problem in MALDI-MS.9 Thus, there is a great need for reducing spatial heterogeneities in MALDI-MS dried droplet applications.
Hydrodynamic flows in the sample droplet play an important role in determining the spatial distribution of samples prepared with the dried-droplet method.10-12 It was found that the evaporation of solvent induces outward capillary flows within droplets, which are responsible for the ring-stain formation.7,10 In contrast, recirculation flows induced by tangential surface-tension gradients may counterbalance the outward capillary flows.13 If the recirculation flow speeds are higher than that of the outward capillary flows, the samples can be efficiently redistributed to reduce the heterogeneity problem.14
In this work, we demonstrate a detailed protocol for preparing samples with a simple drying chamber to induce efficient recirculation flows during droplet drying processes. Droplet drying conditions are precisely controlled, including the temperatures of the sample plate and its surroundings, and the relative humidity within the chamber. The model analytes include maltotriose and bradykinin chain (1-7). The matrix used for the demonstration is 2,4,6-trihydroxyacetophenone (THAP). The samples are examined with time-of-flight (TOF) MS, and the data are analyzed quantitatively to show the reduction of heterogeneity.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Reagent</td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Detergent powder</td>
			<td style="width:157px;">Alconox</td>
			<td style="width:139px;">242985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Methanol</td>
			<td style="width:157px;">Merck</td>
			<td style="width:139px;">106009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Acetonitrile</td>
			<td style="width:157px;">Merck</td>
			<td style="width:139px;">100003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">2,4,6-trihydroxyacetophenone (THAP)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">T64602&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bradykinin fragment (1-7)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">B1651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Maltotriose</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">47884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette tips</td>
			<td style="width:157px;">Mettler Toledo</td>
			<td style="width:139px;">17005091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microcentrifuge tube</td>
			<td style="width:157px;">Axygen</td>
			<td style="width:139px;">MCT-150-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Milli-Q water purification system</td>
			<td style="width:157px;">Millipore</td>
			<td style="width:139px;">ZMQS6VFT1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powder-free nitrile gloves</td>
			<td style="width:157px;">Microflex</td>
			<td style="width:139px;">SU-690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">600 mL beaker</td>
			<td style="width:157px;">Duran</td>
			<td style="width:139px;">2110648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ultrasonic cleaner</td>
			<td style="width:157px;">Delta</td>
			<td style="width:139px;">DC300H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hygrometer</td>
			<td style="width:157px;">Wisewind</td>
			<td style="width:139px;">5330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nitrogen gas flowmeter</td>
			<td style="width:157px;">Dwyer</td>
			<td style="width:139px;">RMA-6-SSV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">K-type thermocouples</td>
			<td style="width:157px;">Digitron</td>
			<td style="width:139px;">311-1670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Centrifuge</td>
			<td style="width:157px;">Select BioProducts</td>
			<td style="width:139px;">Force Mini&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette</td>
			<td style="width:157px;">Rainin</td>
			<td style="width:139px;">pipet-lite XLS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Stereomicroscope</td>
			<td style="width:157px;">Olympus</td>
			<td style="width:139px;">SZX16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Temperature controllable drying chamber</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Synchronized dual-polarity time-of-flight imaging mass spectrometer (DP-TOF IMS)</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">MALDI-TOF stainless steel sample target</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of homogeneous maldi samples for quantitative applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

明視野画像並びにサンプルプレート5の温度25℃で調製マルトトリオース及びブラジキニンフラグメント（1-7）のMS画像は、図1に示されている。ナトリウムイオン付加マルトトリオース、イオン信号主としてを移入の場合それは、25℃のサンプルプレートの温度を用いて調製された試料領域の周囲に。 5℃にサンプルプレートの温度を下げることにより、信?...

Watch this Scientific Journal Video about Preparation of Homogeneous MALDI Samples for Quantitative Applications at JoVE.com

Preparation of Homogeneous MALDI Samples for Quantitative Applications

サンプルの乾燥工程時の基板温度を調節することにより、MALDI質量分析法におけるイオン信号の空間的な不均一性を低減するためのプロトコルが示されています。

定量的なアプリケーションのための均質なMALDI試料の調製

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

preparation-of-homogeneous-maldi-samples-for-quantitative-applications

Research

JoVE Journal

Biochemistry

8.7K ビュー.  Academia Sinica. サンプルの乾燥工程時の基板温度を調節することにより、MALDI質量分析法におけるイオン信号の空間的な不均一性を低減するためのプロトコルが示されています。 

ビデオ: Preparation of Homogeneous MALDI Samples for Quantitative Applications

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

非コード小型RNA定量化のための高密度リポタンパク質の単離

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

生化学

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

細胞培養用ヒドロゲルの蛋白質ベースのアーキテクチャの簡単な操作

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

ペプチド ベースのマトリックスのためにサンプルを固定ホルマリンの最適な準備支援レーザー脱離イオン化質量分析イメージング ワークフロー

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

校正と単一のヌクレオチドの拡張子と MALDI-TOF 質量分析を用いた DNA 修復アッセイ

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

微生物タンパク質および特殊代謝産物データの分析にオープンソースのMALDI TOF-MS IDBacパイプラインを使用

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

タンパク質-脂質相互作用を測定するためのマイクロスケール熱泳動の使用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

液体クロマトグラフィーとタンデム質量分析法を用いたサッカロミセス・セレビシエ細胞リピドームの定量的解析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

並列反応モニタリングによるユビキチン連鎖解析

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

液体クロマトグラフィーとタンデム質量分析法を用いたサッ カロミセス・セレビシエ の定量的メタボロミクス

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

DNA上に集合したEWS-FLI1凝縮体の1分子イメージング

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...