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).

Hinweis: Dieses Protokoll zur Reduzierung der räumlichen Heterogenität Maltotriose und Bradykinin-Fragment (1-7) hergestellt, mit dem getrockneten Tröpfchen Verfahren entwickelt wird. Das Protokoll besteht aus drei Hauptschritte, einschließlich der Vorbereitung und Präkonditionierung, Probenablage und Trocknung und Massenspektrometrie Datenanalyse. Die Verfahren werden beschrieben und im Folgenden näher beschrieben: 1. Vorbereitung und Präkonditionierung <ol><li> Reinigen der Probenplatte <ol><li> Tragen Sie Handschuhe aus Nitril und Hand waschen Sie die Probenplatte vorsichtig mit Reinigungsmittel und destilliertem entionisiertem Wasser (DDW). </li><li> Spülen Sie die Probenplatte mit Methanol (MeOH) und DDW. </li><li> Legen Sie die Probenplatte in einem 600-ml-Becher und füllen mit DDW. </li><li> Beschallen die Probenplatte in DDW für 15 min in einem Ultraschallbad (200 W, 40 kHz). </li><li> Entfernen DDW aus dem Becher und füllen Sie den Becher mit MeOH. </li><li> Beschallen die Probenplatte in MeOH für 15 min im Ultraschallbad (200 W, 40 kHz). </li><li> Brennen Sie die Lösungsmitteltropfen auf der Platte mit Stickstoffgas ab und halten Sie die Probenplatte trocken vor der Probenabscheidung. </li></ol></li><li> Die Regulierung Trockenkammer Temperatur . HINWEIS: Die Trockenkammer ist ein 35 x 20 x 45 cm 3 (B x T x H) Acryl-Kammer Abbildung 1 zeigt das Bild dieses Trocknungssystems. Die Kammer wird mit Raumtemperatur Stickstoffgas durch einen Gasdurchflussmesser bei einer konstanten Strömungsgeschwindigkeit gespült eine niedrige relative Feuchtigkeitsbedingung durch eine geeichte Hygrometer innerhalb der Trocknungskammer eingebaut überwacht aufrechtzuerhalten. Ein Kupferbasisblock in der Trocknungskammer mit einer programmierten konstanten Temperatur Wasser Zirkulator ausgestattet wird verwendet Edelstahlprobenplatten aufzunehmen. Die Kupferbasisblock kann die Probenplatte Temperatur 5-25 ° C zu regulieren. Die Temperaturen der Luft, Kupferbasisblock und der Probenplatte werden durch K-Typ-Thermoelemente überwacht. <ol><li> Öffnen Sie die Tür und legte schnell die Probenplatte auf dem KupferBasisblock schließen Sie die Tür. </li><li> Manuelle Einstellung der Gasdurchflussmesser der Stickstoffvolumenstrom bis 10 Standard-Kubikfuß pro Stunde (SCFH) einzustellen. </li><li> Überwachung der relativen Feuchtigkeit in der Trockenkammer durch das Hygrometer und Feinabstimmung der Gasdurchflussmesser die relative Feuchtigkeit ist immer unter 25% zu gewährleisten. </li><li> Überwachen Sie die Temperatur der Probenplatte durch K-Typ-Thermoelemente und stellen Sie die Wasser Zirkulator Temperatur manuell, bis die Probenplatte erreicht 5 ° C für das Experiment oder Raumtemperatur (25 ° C) für die Steuerung. HINWEIS: Um die Probenplatte bei einer Temperatur ausgelegt, die Wassertemperatur Zirkulator wird typischerweise 0 bis 5 ° C niedriger als die vorgesehene Probe eingestellt zu stabilisieren. Beispielsweise 5 ° C an der Probenplatte zu erhalten, ist die Temperatureinstellung des Wassers Zirkulators im Bereich von 0 bis 2 ° C; der Probenplatte bei 25 ° C, die Temperatureinstellung des Wassers Zirkulator liegt im Bereich von 23 bis 25 ° C zu halten. </li><li> Achten Sie darauf , die erforderlichen Temperaturen und die relative Luftfeuchtigkeit erreicht werden (Tabelle 1) vor der Probenabscheidung. HINWEIS: Alle Parameter sowie deren Einstellwerte für die Trocknungsverfahren mit unterschiedlichen Probenplattentemperaturen sind in Tabelle 1 gezeigt. HINWEIS: Bei einer niedrigen Probenplattentemperatur, Wasserkondensation auf der Probenplatte auftreten können, wenn die Kammertür für eine lange Zeit offen ist. Wenn Wasserkondensation auftritt, schließen Sie die Tür und DO Anzahlung nicht jede Probe auf, bis Kondensation von Wasser ausgetrocknet ist. </li></ol></li><li> Herstellung von Matrix und Analytlösungen <ol><li> Herstellung von Matrixlösungen <ol><li> Bereiten 0,1 M THAP-Lösung mit 50% Acetonitril (ACN): 50% wässrige Lösung DDW. </li></ol></li><li> Präparation von Analyten <ol><li> Bereiten 10 -4 M Maltotriose Lösung mit DDW. </li><li> Bereiten Sie 10 -5 M Bradykinin - Fragment (1-7) Lösung in 50% Acetonitril(ACN): 50% wässrige Lösung DDW. </li></ol></li></ol></li></ol> 2. Probenabscheidung und Trocknung <ol><li> Premix 0,25 ul 0,1 M THAP Lösung und 0,25 ul 10 -4 M Maltotriose oder 10 -5 M Bradykinin - Fragment (1-7) Lösungen in einem Reaktionsgefäß. </li><li> Vortex die gemischte Lösung für 3 Sekunden. </li><li> Zentrifugieren der gemischten Lösung für 2 sec (2,000 xg), um die Lösung am Boden des Zentrifugenröhrchens zu sammeln. </li><li> Öffnen Sie die Tür der Trocknungskammer, deponieren sorgfältig 0,1 ul der Lösung auf der Probenplatte mit Pipette und schließen Sie sofort die Tür. </li><li> Warten Sie auf die Probe Tröpfchen zu trocknen. . HINWEIS: Die typischerweise beobachtet Trocknungszeiten mit unterschiedlichen Probenplattentemperaturen in aufgeführt sind Tabelle 1 Für die Probenplattentemperatur von 5 ° C, die durchschnittliche Trocknungszeit beträgt 800 bis 1000 sec; für die Probenplattentemperatur von 25 ° C beträgt die durchschnittliche Trocknungszeit 100 bis 150 sec. </li><li> Nach dem Trocknen die Tür öffnen, der Trocknungskammer. </li><li> Stellen Sie den Wasser Zirkulator Temperatur auf Raumtemperatur (25 ° C). HINWEIS: Überspringen Sie diesen Schritt, wenn die Probenplatte ständig bei Raumtemperatur (25 ° C) während des Trocknungsprozesses gehalten wird. </li><li> Nach der Probenplatte Temperatur wieder auf Raumtemperatur (25 ° C), Entfernen von der Trocknungskammer, um die Probenplatte. </li><li> Untersuchen Sie die Probenmorphologie unter einem 5fach-Stereomikroskop und einen Schnappschuss Hellfeldbild. HINWEIS: Wenn die Kristallmorphologien nicht erwartet werden, ist es notwendig, eine neue Probe mit dem gleichen Verfahren herzustellen. Typische Kristallmorphologien sind in den oberen Platten der Figur 2 gezeigt. HINWEIS: In den Fällen mit geringen Probenplattentemperaturen, wie 5 ° C, es ist wichtig, die Probenplatte auf Raumtemperatur aufwärmen, bevor es aus der Trocknungskammer nehmen. Wenn die Proben Ablagerung, halten Sie nicht die vorgemischten solution in der Spitze der Pipette über 10 sec. Sie nicht die vorgemischten Lösung wieder verwenden , nachdem die Proben abgeschieden werden . Die oberen Platten von 2 zeigen Hellfeld - Bilder von Proben hergestellt mit unterschiedlichen Probenplattentemperaturen. </li></ol> 3. Mass Spectrometry Datenanalyse <ol><li> Mass Spectrometry Datenerfassung HINWEIS: Nach der Zubereitung kann die Probe mit bildgebenden Massenspektrometrie analysiert werden. In der aktuellen Studie werden die Imaging - MS - Experimente durchgeführt , ein Labor gebauten Synchronized Dual-Polarität TOF mit (DP-TOF) Imaging - Massenspektrometer. 15 Handels MALDI-TOF - Massenspektrometer mit Imaging - Fähigkeit eignen sich auch für solche Experimente. Das Massenspektrometer wird in linearer Extraktion und positive Ionen-Modi mit optimierter Extraktions Verzögerungen betrieben. Die kinetische Energie der Ionen beträgt 20 kV. Der Laserstrahl Größe beträgt 35 &amp; mgr; m im Durchmesser auf der Probenoberfläche, und das Spektrum von jedem Spot ist der aveWut von 5 Laserschüssen. <ol><li> Legen Sie die Probenplatte in das MALDI-Massenspektrometer. </li><li> Führen Sie die Abbildung Massenspektrometrie-Analyse der Probe in Schritten hergestellt 2,1-2,9. </li><li> Wählen Sie eine charakteristische Massenspitze aus der Massenliste im Ergebnisfenster angezeigt und klicken Sie auf &quot;2D&quot;, um ein zweidimensionales Ionenbild zeichnen. HINWEIS: Bei Maltotriose gemischt mit THAP die charakteristischen Spitzen sind sodiated Maltotriose, protonierten THAP und sodiated THAP. Für Bradykinin-Fragment (1-7) mit THAP gemischt, umfassen die charakteristischen Peaks Bradykinin Fragment protoniert (1-7), protonierten THAP und sodiated THAP. </li><li> Klicken Sie auf die Tasten zur Einstellung der im Popup-Fenster die oberen und unteren Grenzen der Signalintensität zu bestimmen, und klicken Sie auf &quot;Bild speichern&quot;. Diese Einstellung definiert den Kontrast von Ionen Bilder. HINWEIS: In jedem einzelnen Satz von Daten, die gerissenen Bereiche und die Null-Punkte niedriger Helligkeit eliminiert zeigen. </li><li> Bitte beachten und zu vergleichen, das IonBild mit dem Hellfeld-Bild, das in Schritt 2.9 gemacht. HINWEIS: Imaging-Massenspektrometrie und Konstruktion von Bildern von bestimmten Ionen können mit kommerziellen Geräten erreicht werden. Aufgrund der Vielzahl der Datenerfassung und Analyse-Software, sollten die Anwender Software-Anweisungen vom Instrument Anbieter bereitgestellten folgen Bilder von hoher Qualität zu erhalten. </li></ol></li><li> Datenanalyse HINWEIS: Die Heterogenität der Proben quantitativ analysiert. In dieser Demonstration wird jede Probe von Software im eigenen Haus entwickelt in mehrere konzentrische Bereiche unterteilt, die räumliche Verteilung der Ionen zu analysieren. Die Analyse kann auch eigenständige Datenanalyse-Software durchgeführt werden. <ol><li> Klicken Sie auf die Nullstellen und die gerissenen Bereiche in dem Ionenbild im Ergebnisfenster angezeigt zu unwichtigen Bereichen zu entfernen. Hinweis: Dieses Verfahren definiert die wesentlichen Bereich des Ionenbild. </li><li> Klicken Sie die Schaltfläche &quot;Kante finden&quot;, um die äußerste Schicht des Ionenbild zu finden. </li><li> Klicken Sie auf &quot;abziehen&quot;, um den Ionenfluss Informationen der äussersten Schicht in einer Datenbank zu speichern und diese Schicht aus dem Ionen Bild entfernen gleichzeitig. Ein Kontrollkästchen diese äußerste Schicht darstellt, wird in der &quot;Ausgangsdaten&quot; Liste des Ergebnisfensters angezeigt. </li><li> Wiederholen Sie die Schritte 3.2.2 und 3.2.3 bis das Zentrum des Ionenbild definiert ist. </li><li> Klicken Sie auf und wählen Sie alle Kontrollkästchen in der &quot;Ausgangsdaten&quot; und klicken Sie auf &quot;Export&quot;, um die Daten zu exportieren. </li><li> Öffnen Sie die exportierten Daten Tabellenkalkulations-Software mit dem durchschnittlichen Ionenhäufigkeit jeder Schicht zu berechnen, die räumliche Verteilung Informationen von Ionen zu erhalten. </li></ol></li></ol>

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The authors declare no competing financial interest.

Basierend auf früheren theoretischen Vorhersagen, temperaturbedingten hydrodynamischen Strömungen innerhalb Tröpfchen nach außen Kapillare fließt überwinden durch Verdampfen des Lösungsmittels induziert. Die Effizienz solcher interne Rezirkulation von Molekülen wird erhöht, wenn die Temperatur innerhalb eines Tröpfchens Zunahme Steigungen. Entsprechend den vorhergesagten Ergebnissen, wenn die Probenplatte Temperatur unter 5 ° C gehalten wurde, während seine Umgebung bei Umgebungstemperatur gehalten wird, ist...

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 border="0" cellpadding="0" cellspacing="0" width="787">
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		<col />
	</colgroup>
	<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.

Die Hellfeld - Bilder sowie die MS Bilder von Maltotriose und Bradykinin - Fragment (1-7) hergestellt , mit Probenplattentemperatur von 5 und 25 ° C sind in 1 gezeigt. Im Fall der sodiated Maltotriose, das Ionensignal hauptsächlich bevölkert an der Peripherie des Gebietes Probe, wenn es mit einer Probe Plattentemperatur von 25 ° C hergestellt. die Probenplatte Temperatur auf 5 ° C, füllt das Signal homogen über die gesamte Probenfläche Durch die Verringerung. Der...

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

Ein Protokoll für die räumliche Heterogenitäten der Ionensignale in der MALDI-Massenspektrometrie Reduzierung von Substrattemperatur während der Probentrocknungsprozesse regulieren demonstriert.

Herstellung von homogenen MALDI Proben für die quantitative Anwendungen

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 Aufrufe.  Academia Sinica.  Ein Protokoll für die räumliche Heterogenitäten der Ionensignale in der MALDI-Massenspektrometrie Reduzierung von Substrattemperatur während der Probentrocknungsprozesse regulieren demonstriert. 

Serie: 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.

Isolierung von Lipoproteinen hoher Dichte für 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 ...

Forschung

Biochemie

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.

Einfache Handhabung von Architekturen in Protein-basierte Hydrogele für Zelle Kultur Anwendungen

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.

Optimale Vorbereitung von Formalin fixiert Proben für Peptid-Matrix basierten Assisted Laser Desorption/Ionisierung Massenspektrometrie Imaging-Workflows

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.

Korrekturlesen und DNA-Reparatur-Assay mit Einzel-Nukleotid-Erweiterung und MALDI-TOF Massenspektrometrie-Analyse

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.

Verwendung der Open-Source MALDI TOF-MS IDBac Pipeline zur Analyse von mikrobiellen Protein- und Spezialisierten Metabolitendaten

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.

Einsatz der mikroskaligen Thermophorese zur Messung von Protein-Lipid-Wechselwirkungen

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.

Quantitative Analyse des Zelllipidoms von Saccharomyces Cerevisiae mit Flüssigchromatographie gekoppelt mit Tandem-Massenspektrometrie

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.

Ubiquitin-Kettenanalyse durch Parallelreaktionsüberwachung

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.

Quantitative Metabolomik von Saccharomyces Cerevisiae mittels Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie

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.

Einzelmolekül-Bildgebung von EWS-FLI1-Kondensaten auf 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 ...