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.

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

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

フライトのマトリックス支援レーザー脱離/イオン化飛行時間（MALDI-TOF）100kDaのより大きなインタクトタンパク質の質量分析

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

化学

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

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

バイオマーカー探索のためのメソポーラスシリカ薄膜の低分子量蛋白質の濃縮

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

生物工学

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 ...

生化学

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

サイズ排除ICP-MSを用いた定量Metalloproteomic分析のための基準

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

全細胞MALDI-TOF質量分析は、マクロファージ活性化の異なるモードを分析するための正確かつ迅速な方法である

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

免疫・感染学

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

マトリックス支援レーザー脱離イオン化質量分析法における炭水化物イオン信号を強化する効率的なサンプル調製法

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

変換にマトリックス支援レーザー脱離/イオンイメージングのためのマトリックスとしてジスラノールはイオンサイクロトロン共鳴質量分析計をフーリエ

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

代謝産物の調査のためのMALDI-質量分析イメージング<em&gt;ウマゴヤシtruncatula</em&gt;ルート結節

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

MALDI質量分析イメージングを用いた代謝プロファイリングのサンプル調製

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

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

要約

さらに動画を探す

フライトのマトリックス支援レーザー脱離/イオン化飛行時間（MALDI-TOF）100kDaのより大きなインタクトタンパク質の質量分析

MALDIサンプル調製：超薄層法

バイオマーカー探索のためのメソポーラスシリカ薄膜の低分子量蛋白質の濃縮

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

サイズ排除ICP-MSを用いた定量Metalloproteomic分析のための基準

全細胞MALDI-TOF質量分析は、マクロファージ活性化の異なるモードを分析するための正確かつ迅速な方法である

マトリックス支援レーザー脱離イオン化質量分析法における炭水化物イオン信号を強化する効率的なサンプル調製法

変換にマトリックス支援レーザー脱離/イオンイメージングのためのマトリックスとしてジスラノールはイオンサイクロトロン共鳴質量分析計をフーリエ

代謝産物の調査のためのMALDI-質量分析イメージング

MALDI質量分析イメージングを用いた代謝プロファイリングのサンプル調製

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

要約

さらに動画を探す

フライトのマトリックス支援レーザー脱離/イオン化飛行時間（MALDI-TOF）100kDaのより大きなインタクトタンパク質の質量分析

MALDIサンプル調製：超薄層法

バイオマーカー探索のためのメソポーラスシリカ薄膜の低分子量蛋白質の濃縮

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

サイズ排除ICP-MSを用いた定量Metalloproteomic分析のための基準

全細胞MALDI-TOF質量分析は、マクロファージ活性化の異なるモードを分析するための正確かつ迅速な方法である

マトリックス支援レーザー脱離イオン化質量分析法における炭水化物イオン信号を強化する効率的なサンプル調製法

変換にマトリックス支援レーザー脱離/イオンイメージングのためのマトリックスとしてジスラノールはイオンサイクロトロン共鳴質量分析計をフーリエ

代謝産物の調査のためのMALDI-質量分析イメージング

MALDI質量分析イメージングを用いた代謝プロファイリングのサンプル調製

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