Diese Arbeit wurde von der Energy Biosciences Institute gewähren OO0G01 finanziert.

Die OLIMP Methode beruht auf der großen Hemizellulose in den Zellwänden von zweikeimblättrigen Pflanzen, Xyloglucan Beispiel mit Hilfe eines Endoglucanase als glycosylhydrolase. Die Methode ist mit ganzen Arabidopsis-Keimlinge als Pflanzengewebe Quelle demonstriert. Enzyme und extrazelluläre Matrix-Material kann ersetzt je nach gewünschter Analyse mit dem gleichen Verfahren werden. 1. Zellwand isoliert <ol><li> Ernte fünf 5-Tage alte gesamten Arabidopsis-Keimlinge oder den entsprechenden Betrag von Ihrem gewünschten Material und übertragen Sie sie in ein 1,5 ml Reaktionsgefäß. Stellen Sie sicher, dass das Material in den Boden des Röhrchens befindet. </li><li> Fügen Sie zwei 3mm Metallkugeln in das Reaktionsrohr auf der Probe und Snap-in flüssigem Stickstoff einfrieren. </li><li> Grind die gefrorene Probe mit einer Kugelmühle (2:30 min, 25 Hz). </li><li> 1ml 70% wässrigem Ethanol zur Probe. Entfernen Sie die Metallkugeln mit einem Magneten. Vortex gründlich. </li><li> Centrifuge Probe bei 14.000 rpm für 10 Minuten, um Pellets der Alkohol unlösliche Rückstand enthält die Zellwand Material. </li><li> Entfernen Sie vorsichtig den Überstand durch Absaugen. Achten Sie darauf, das Pellet stören. </li><li> 1ml von Chloroform / Methanol (1:1 v / v) Lösung. Vortex gründlich, um das Pellet zu resuspendieren. </li><li> Centrifuge Probe bei 14.000 rpm für 10 min und entfernen Sie vorsichtig den Überstand durch Absaugen. Achten Sie darauf, das Pellet stören. </li><li> Trocknen Sie die Probe mit einem Konzentrator. </li></ol> 2. Solubilisierung von Oligosacchariden <ol><li> Für die Solubilisierung der jeweiligen Polysaccharid, das in Form seines Oligosaccharide resuspendieren die trockene Pellet in 25 &amp;mgr; l eines geeigneten Puffers. Für die Endoglucanase 50mM Ammoniumformiat, pH4.5, verwendet wird. Add 0.2U der Endoglucanase. Vortex die Aussetzung und Spin Flüssigkeit nach unten. </li><li> Inkubieren Sie die Probe für 16h bei 37 ° C in einem Inkubator, Schütteln bei 300rpm. </li><li> Entfernen des Lösungsmittels der Verdauung (Wasser) mit einem Konzentrator. </li></ol> 3. MALDI-TOF-Analyse des freigesetzten Oligosaccharide <ol><li> Biorex MSZ 501 Kationenaustauscherharz Perlen werden verwendet, um Salze und Enzym aus der verdauten Probe zu entfernen. Zustand der Perlen, indem ein Aliquot in eine leere Spalte und Waschen der Beads mit reichlich Wasser. </li><li> Add 5-10 Biorex Perlen, die verdaut und getrocknete Probe. Dann fügen Sie 10 &amp;mgr; l Wasser, für kleinere Mengen an Material 5μl von Wasser verwendet werden können. Tauchen Sie die Perlen in der Lösung durch kurzes Drehen der Röhre. Inkubieren für mindestens 10 Minuten bei Raumtemperatur. </li><li> Spot 2μl Matrix (2,5-Dihydroxybenzoesäure (DHB), 10mg/ml in Wasser) auf ein MALDI Zieltafel. Man dampft die Matrix Lösungsmittel unter Vakuum führt zu homogenen Matrix chemische Kristalle. </li><li> Spot 2μl der entsalzt Probelösung auf der Matrix-Kristalle auf die Zielscheibe. Wenn Sie eine große Anzahl von Proben vor Ort wollen nur an der Erkennung von maximal 3min zu halten. Dieser Zeitrahmen ist sichergestellt, dass der erste entdeckte Probe nicht trocken ist, noch. Warten Sie eine zusätzliche 2min zu einer Durchmischung wieder aufgelöst Matrix und Probe Oligosaccharidmoleküle in den letzten spotted Probe zu gewährleisten. Dann trocknen die Zielscheibe in einem Vakuum. Die Probe / Matrix-Mischung sollte in weniger als 1 Minute, um eine homogene Kristallisation ermöglichen kristallisieren. </li><li> Legen Sie Zieltafel in ein MALDI-TOF-Massenspektrometer. Die Maschine sollte auf positive Reflektron-Modus mit einer Beschleunigungsspannung von 20000V und einer Verzögerung von 350 nm eingestellt werden; die ausgewählten Massenbereich sollte 500 bis 3000 Da werden (kann sich ändern, abhängig von der erwarteten Oligomere). Starten Auslösen des Laserstrahls und sammeln 100-200 Spektren pro Probe, die kompiliert werden, um einen repräsentativen Durchschnitt Spektrum (Abbildung 2A) zu erzeugen. Ionen repräsentieren spezifische Oligosaccharide können durch ihre Masse über Ladung (m / z)-Verhältnis ermittelt werden. Die Ionenintensitäten (Peakhöhe) aller Ionen von Interesse sollten bis zu 100%, was die relative Häufigkeit der einzelnen Oligosaccharid in der Probe (Abbildung 2B) hinzugefügt werden. </li></ol> 4. In situ OLIMP Analyse OLIMP können auch direkt auf das Gewebe Weglassen jeder Wand Vorbereitungsschritte eingesetzt werden. Als Beispiel etiolierten Hypokotyledonen Arabidopsis als Pflanzengewebe Quelle verwendet werden. Auch hier ist eine Endoglucanase verwendet werden, um die Struktur der Hemicellulosen Xyloglucan zu bestimmen. Enzyme und Gewebe Material ersetzt je nach gewünschter Analyse mit dem gleichen Verfahren werden. <ol><li> Ernten im etiolierten Keimling und legen Sie es auf eine leere MALDI Zieltafel und lassen Sie die Sämlinge trocken. </li><li> Add 0.5μl Endoglucanase (0.4U/μl, in 50 mM Ammoniumformiat aufgelöst, pH4.5) auf die getrocknete Keimling an der gewünschten Position. Stellen Sie sicher, dass das Enzym drop teilweise berührt die Zieltafel. </li><li> Legen Sie die MALDI Zieltafel in einem geschlossenen Behälter mit sättigenden Feuchtigkeit zu vermeiden, dass das Enzym drop austrocknet. Die Platte in die Kammer für 16h bei 37 ° C. </li><li> Dry das MALDI-Target-Platte mit dem Pflanzengewebe und das Enzym drop unter Vakuum. </li><li> Add 0.5μl Matrix (DHB; 10mg / l) auf der jeweils getrocknet Enzym vor Ort. Lassen Sie es für 2min stehen. </li><li> Trocknen Sie die MALDI Zieltafel unter Vakuum. </li><li> Analysieren Sie die Proben mit der MALDI-TOF. Legen Sie Zieltafel mit dem Gewebe und das Enzym / Matrix-Spot (s) in ein MALDI-TOF-Massenspektrometer. Die Maschine sollte auf positive Reflektron-Modus mit einer Beschleunigungsspannung von 20000V und einer Verzögerung von 350 nm eingestellt werden; die ausgewählten Massenbereich sollte 500 bis 3000 Da werden (kann sich ändern, abhängig von der erwarteten Oligomere). </li><li> Starten Auslösen des Laserstrahls auf die Matrix / Probe-Kristalle NEXT, um das Gewebe. Stellen Sie sicher, daß Sie nicht treffen das Gewebe selbst, wie es in einem solchen Fall die Zeit des Fluges der Moleküle wird ausgeschaltet. Sammeln Sie ca. 20-50 Spektren pro Spot, der kompiliert werden, um einen repräsentativen Durchschnitt Spektrum (Abbildung 3) zu erzeugen. Ionen repräsentieren spezifische Oligosaccharide können durch ihre Masse über Ladung (m / z)-Verhältnis ermittelt werden. Die Ionenintensitäten (ion Höhe) gemeldet und alle Ionen von Interesse sollten bis zu 100%, was die relative Häufigkeit der einzelnen Oligosaccharide in der Probe zugegeben werden. </li></ol> 5. Repräsentative Ergebnisse Ein Beispiel für eine OLIMP Analyse der Hemicellulosen Xyloglucan in Arabidopsis-Keimlinge ist in Abbildung 2 dargestellt. Aufgrund der Masse Unterschiede der Ionen und der bekannten gut charakterisierte Enzym (Endoglucanase) Spezifität der Ionen kann auf bestimmte Oligosaccharid-Strukturen (Abbildung 2A) zugeordnet werden. Offensichtlich kann Strukturisomeren nicht unterschieden werden. Die Grundannahme für die Bestimmung der relativen Häufigkeit der verschiedenen Oligosaccharide (Abbildung 2B) ist, dass ihre Massenspektrometrie Responsefaktor sehr ähnlich für die Oligosaccharide ist. Wie hier gezeigt, ist die OLIMP Quantifizierung hoch reproduzierbar. Beachten Sie jedoch, dass Robustheit des Verfahrens stark abhängig von der Signal-Rausch-Verhältnis der verschiedenen Oligosaccharid-Ionen ist. Zum Beispiel Verunreinigungen mit Salzen oder geringere Mengen von Oligosacchariden abnehmen kann dieses Verhältnis. Die OLIMP Analyse auf das Gewebe selbst (in-situ-Analyse) ermöglicht die Untersuchung von sehr kleinen und definierten Bereichen und beinhaltet sehr wenig Probenvorbereitung. In dem hier vorgestellten Beispiel (Abbildung 3) keine qualitativen (gleiche Ionen), aber quantitative Unterschiede (Variation in der Ionenintensitäten) wurden zwischen dem Spross und Wurzel-Gewebe des Arabidopsis Sämling beobachtet. Permutationen der OLIMP-Methode könnte somit das Gewebe &quot;imaging&quot; führen. <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Flussdiagramm der OLIMP Verfahren mit ganzen Arabidopsis-Keimlinge als einer pflanzlichen Quelle. Zuerst wird das Gewebe mazeriert und Zellwand Material hergestellt (Bild aus Fujino et al modifiziert. 10). Dann Oligosaccharide einer bestimmten Wand Polysaccharid freigesetzt werden unter Verwendung eines spezifischen Hydrolase. Schließlich werden die relativen Häufigkeiten der löslichen Oligosacchariden bestimmt mittels MALDI-TOF-Massenspektrometrie. <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Relative Häufigkeit von Xyloglucan Oligosaccharide in etiolierten Keimlingen von Arabidopsis als von OLIMP bestimmt. A) Repräsentative Xyloglucan Oligosaccharid Massenspektrum; jedes Ion stellt eine spezifische Oligosaccharid-Struktur kann Strukturisomeren nicht unterschieden werden. B) Entsprechende Balkendiagramm für die Bestimmung der relativen Oligosaccharid Hülle und Fülle. <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="Abbildung 3" /> Abbildung 3. In situ OLIMP Analyse mit etiolierten Keimlingen von Arabidopsis als Beispiel. Jedes Enzym / Verdauung Tröpfchen (farbige Kreise) können unabhängig voneinander analysiert werden und eine entsprechende Massenspektren erhalten und analysiert werden. <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="Abbildung 4" /> Abbildung 4. OLIMP Spektrum der Hemizellulose Xylan durch Verdauen von Miscanthus Blattmaterial mit einer Xylanase (Megazyme) erhalten.

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+xylan+synthesis+mutants+reveals+new+insight+into+the+mechanisms+of+xylan+synthesis.">Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis.</a> Plant Journal. 52 (6), 1154-1154 (2007).</li><li>Lee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PARVUS+gene+is+expressed+in+cells+undergoing+secondary+wall+thickening+and+is+essential+for+glucuronoxylan+biosynthesis.">The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis.</a> Plant Cell Physiol. 48 (12), 1659-1659 (2007).</li><li>Obel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectin+may+hinder+the+unfolding+of+xyloglucan+chains+during+cell+deformation:+implications+of+the+mechanical+performance+of+Arabidopsis+hypocotyls+with+pectin+alterations.">Pectin may hinder the unfolding of xyloglucan chains during cell deformation: implications of the mechanical performance of Arabidopsis hypocotyls with pectin alterations.</a> Molecular Plant . , (2009).</li><li>Cavalier, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+irregular+xylem9+mutant+is+deficient+in+xylan+xylosyltransferase+activity.">The irregular xylem9 mutant is deficient in xylan xylosyltransferase activity.</a> Plant and Cell Physiology. 48 (11), 1624-1624 (2007).</li><li>Iglesias, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+glycosidases+active+against+xyloglucan+oligosaccharides+of+Arabidopsis+thaliana.">Apoplastic glycosidases active against xyloglucan oligosaccharides of Arabidopsis thaliana.</a> Plant and Cell Physiology. 47 (1), 55-55 (2006).</li><li>Fujino, T., Sone, Y., Mitsuishi, Y., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cross-links+between+cellulose+microfibrils,+and+their+occurrence+during+elongation+growth+in+pea+epicotyl.">Characterization of cross-links between cellulose microfibrils, and their occurrence during elongation growth in pea epicotyl.</a> Plant Cell Physiol. 41 (4), 486-486 (2000).</li><li>Vanzin, G. 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Die OLIMP hier vorgestellte Methode ermöglicht eine sehr sensible und schnelle Analyse von Polymeren, die in der extrazellulären Matrix. OLIMP verbindet die enzymatische Freisetzung von Oligomeren mit anschließender MALDI-TOF-Analyse. Die Erzeugung eines MALDI-TOF-Spektrum dauert weniger als eine Minute, damit OLIMP ist geeignet für eine breite Palette von Anwendungen, einschließlich High-Throughput-Studien wie Mutantenscreens. OLIMP ist nicht auf pflanzliche Polysaccharide beschränkt, sondern kann potenziell ein breites Spektrum von Polymeren, die nur durch die Verfügbarkeit von bestimmten hydrolytische Enzyme beschränkt angewendet werden. Allerdings ist eine Beschränkung von OLIMP, dass eine absolute Fülle des Polymers nicht erreicht werden kann. Wie bereits erwähnt OLIMP kann verwendet werden, um die Struktur einer Vielzahl von Polysacchariden in der extrazellulären Matrix von einer Vielfalt der Arten zu studieren. Als Beispiel stellt Abbildung 4 eine OLIMP Spektrum der wichtigsten Hemizellulose in Grasarten, Xylan. Hier wurde Zellwand Material aus den gemäßigten Gras Miscanthus abgeleitet verdaut mit einer Xylanase.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2,5-dihydroxybenzoic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>37550</td>
<td>10mg/mL in water</td>
</tr>
<tr>
<td>BioRex MSZ 501(D) Resin</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>142-7425</td>
<td>&nbsp;</td>
<tr>
<td>Endoglucanase</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-CELTR</td>
<td>&nbsp;</td>
<tr>
<td>Xylanase M6</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-XYRU6</td>
<td>&nbsp;</td>
<tr>
<td>3mm metal balls</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>22.455.0011</td>
<td>&nbsp;</td>
<tr>
<td>Beat mill</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>Mixer Mill MM400</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>&nbsp;</td>
<td>Shimadzu BioTech</td>
<td>Axima Performance</td>
<td>&nbsp;</td>
<tr>
<td>MALDI target plate</td>
<td>&nbsp;</td>
<td>Kratos Analytical</td>
<td>DE4555TA</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>Vacufuge 5301</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSVMHTS00</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pump</td>
<td>&nbsp;</td>
<td>Welch</td>
<td>DryFast Ultra 2032</td>
<td>&nbsp;</td>		</tbody>
		</table>
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oligo mass profiling (olimp) of extracellular polysaccharides

Der direkte Kontakt der Zellen für die Umwelt ist in vielen Organismen durch eine extrazelluläre Matrix vermittelt. Ein gemeinsamer Aspekt der extrazellulären Matrix ist, dass sie komplexe Zuckerreste in Form von Glykoproteinen, Proteoglykanen und / oder Polysaccharide enthalten. Beispiele hierfür sind die extrazelluläre Matrix des Menschen und tierischen Zellen, die hauptsächlich aus fibrillären Proteinen und Proteoglykanen oder Polysaccharid Zellwände von Pflanzen und Pilzen, und die Proteoglykan / Glycolipid Basis Zellwände von Bakterien. Alle diese Glykostrukturen spielen entscheidende Rollen in Zell-Zell-und Zell-zu-Umfeld Kommunikation und Signalisierung. Eine außerordentliche komplexeres Beispiel für eine extrazelluläre Matrix ist in die Wände der höheren Pflanzenzellen. Ihre Wand ist fast ausschließlich aus Zucker gemacht, bis zu 75% Trockengewicht, und besteht aus den am häufigsten vorkommende Biopolymere präsentieren auf diesem Planeten. Daher ist die Forschung durchgeführt, wie diese Materialien am besten zu nutzen als eine Kohlenstoff-neutrale erneuerbare Ressource zu petrochemischen Produkten aus fossilen Brennstoffen gewonnen zu ersetzen. Die größte Herausforderung für Brennstoff Umwandlung bleibt die Widerspenstigkeit der Wände enzymatische oder chemische Abbau aufgrund der einzigartigen Glykostrukturen in dieser einzigartigen Biokomposit. Hier stellen wir ein Verfahren zur schnellen und empfindlichen Analyse der pflanzlichen Zellwand Glykostrukturen. Diese Methode Oligo Messe Profiling (OLIMP) ist die enzymatische Freisetzung von Oligosacchariden aus Wandmaterialien Erleichterung spezifische Glykosylhydrolasen und anschließende Analyse der gelösten Oligosaccharid Mischungen unter Verwendung von Matrix-Assisted Laser Desorption / Ionisation Time-of-flight Massenspektrometrie (MALDI-TOF/MS Basis ) 1 (Abbildung 1). OLIMP erfordert Wände von nur 5000 Zellen für eine vollständige Analyse können auf das Gewebe selbst 2 durchgeführt werden, und ist zugänglich für High-Throughput-Analysen 3. Während die absolute Menge des gelösten Oligosaccharide können nicht durch OLIMP bestimmt die relative Häufigkeit der verschiedenen Oligosaccharid-Ionen aus den Massenspektren geben Einblicke über die Substitution-Muster der nativen Polysaccharid in der Wand abgegrenzt werden. OLIMP kann verwendet werden, um eine Vielzahl von Wand-Polymeren, die nur durch die Verfügbarkeit von bestimmten Enzymen 4 begrenzt zu analysieren. Zum Beispiel für die Analyse von Polymeren, die in der pflanzlichen Zellwand Enzyme sind für die Hemicellulosen Xyloglucan mit einem Xyloglucanase 5, 11, 12, 13, Xylan mit einem Endo-β-(1-4)-Xylanase 6,7 analysieren oder für Pektinstoffe Polysacchariden mit einer Kombination aus einer Polygalacturonase und Methylesterase 8. Darüber hinaus kann nach den gleichen Grundsätzen von OLIMP glycosylhydrolase und sogar Glycosyltransferase Aktivitäten überwacht und bestimmt werden 9.

Watch this Scientific Journal Video about OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides at JoVE.com

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

Eine schnelle Art und Weise beschrieben, Einblicke in die Struktur der Polysaccharide in einer extrazellulären Matrix zu gewinnen. Die Methode nutzt die Spezifität von Glykosylhydrolasen und die Empfindlichkeit der Massenspektrometrie ermöglicht winzige Mengen von Materialien analysiert werden. Diese Technik ist anpassungsfähig, um direkt auf das Gewebe selbst verwendet werden.

Oligo Messe Profiling (OLIMP) der extrazellulären Polysacchariden

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

oligo-mass-profiling-olimp-of-extracellular-polysaccharides

Research

JoVE Journal

Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

13.6K Aufrufe.  University of California, Berkeley. Eine schnelle Art und Weise beschrieben, Einblicke in die Struktur der Polysaccharide in einer extrazellulären Matrix zu gewinnen. Die Methode nutzt die Spezifität von Glykosylhydrolasen und die Empfindlichkeit der Massenspektrometrie ermöglicht winzige Mengen von Materialien analysiert werden. Diese Technik ist anpassungsfähig, um direkt auf das Gewebe selbst verwendet werden.

Serie: OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

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-Probenvorbereitung: Die Ultra Thin-Layer-Methode

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Forschung

Biologie

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Antikörper Profiling von Luciferase Immunpräzipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profiling von Methyltransferasen und andere S-Adenosyl- L-Homocystein-bindende Proteine ​​von Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Single Cell Transcriptional Profiling der erwachsenen Maus Kardiomyozyten

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Profiling Thiolredox Proteome Mit Isotopen-Massenspektrometrie Tagging

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Glycan Profiling of Plant Cell Wall Polymers mit Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Analyse der Translation Initiation Während Stress-Bedingungen durch Polysomprofile Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Bewertung der Selective mRNA Translation in Säugerzellen durch Polysomen Profilieren

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Anreicherung von extrazellulären Matrixproteinen aus Gewebe und Verdauung in Peptide für Massenspektrometrie-Analyse

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Cellular Redox Profiling mit hochauflösender Mikroskopie

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...