Diese Forschung wurde von der Startfonds der Simon-Fraser-Universität unterstützt.

Ein. Ernte-Membranen <ol><li> Hinzufügen hypotonischen Puffer (10 mM Tris-HCl, 10 mM KCl, 1,5 mM MgCl 2, pH 8,0) mit Proteaseinhibitorcocktail auf einen Zellpellet (~ 10 8 Zellen) und Inkubation für 15-30 min auf Eis. Lyse der Zellen, indem die Probe durch Spritzennadeln (5-10 Durchläufe) oder Homogenisieren der Probe von einem Dounce Homogenisator (15-30 Schläge). Verwenden Sie eine Zählkammer und Trypanblaufärbung um die Effizienz der Lyse zu überprüfen. </li><li> Erhalten Sie die mikrosomale Fraktion durch eine differenzielle Zentrifugation des Lysats bei 3.000 gx 15 min und dann bei 100.000 gx 2 Stunden. Die erste Zentrifugation erhält man ein Pellet, das die Kernfraktion und die anschließende Zentrifugation des Überstandes erzeugt ein zweites Pellet, welches die Mikrosomenfraktion, die die Plasmamembran als auch membrangebundene Organellen 8 enthält. Die endgültige Überstand ist die cytosolische Fraktion. Bewahren Sie die mikrosomalen Fraktionen in -80 ° C-Gefrierschrank für weitere analysist wie nachstehend angegeben. </li></ol> 2. Auflösen, denaturieren und Digest Membranproteine <ol><li> Löse den mikrosomalen Fraktion im Denaturierungspuffer (40 mM Tris, 10 mM EDTA, 10 mM TCEP, 0,5% Rapigest, pH 8) und dann inkubieren der Lösung auf 100 ° C für 10 min. </li><li> Kühlen der erhitzten Lösung auf Raumtemperatur abgekühlt, ultrahochreinen Harnstoffpulver in der Lösung auf 8 M Endkonzentration und Inkubation der Probe bei 37 ° C für 30 min. </li><li> Hinzufügen Iodacetamid Stammlösung zu der Probe auf 15 mM Endkonzentration, und Inkubieren der Lösung in der Dunkelheit für 30 min bei Raumtemperatur, um die freien Thiole in der Probe zu alkylieren. Hinzufügen DTT-Stammlösung zu der Probe auf 10 mM Endkonzentration der Lösung und Inkubation für weitere 10 min bei Raumtemperatur, um das überschüssige Iodacetamid stillen. </li><li> Verdünnt die erhaltene Lösung 10-fach mit 40 mM Tris-Puffer bei pH 8, und anschließend Trypsin hinzufügen, um die Probe bei einer 1:20-Verhältnis von trypsin zum Gesamtprotein. Aufrechterhaltung der Aufschlussreaktion in einem 37 ° C Ofen über Nacht, um sicherzustellen, wird die Reaktion beendet. </li><li> Beenden Sie die Verdauung durch Ansäuern der Probenlösung mit HCl, ein Zustand, bricht auch das Reinigungsmittel, dh Rapigest pH 1. Verschlechtern die Rapigest bei 37 ° C für 1 h, und entfernen Sie die entstandene Niederschlag durch Zentrifugieren. </li><li> Reinigen Sie den Überstand, die Probe-Peptide enthält, die von einem C-18-Festphasenextraktion (SPE) Patrone und trocknen Sie die erhaltene Probe durch Speedvac. </li><li> Durchführen einer SDS-PAGE-Analyse von Proben vor und nach der Trypsin-Verdau, um die Effizienz der Verdauung zu bestätigen. </li></ol> 3. Glycopeptid-Capture- <ol><li> Man löst die gereinigten Peptide im Kopplungspuffer (100 mM Natriumacetat, pH 5,5). Hinzufügen Natriumperiodat in die Peptidlösung in einer Endkonzentration von 10 mM für 30 min im Dunkeln bei Raumtemperatur. Dadurch werden die cis-Diol-Gruppen in den Glycanen zu Aldehyden oxidieren, Welche die Glykane zu koppeln Harzes durch Hydrazid Chemie ermöglichen. Quenche die übermäßige Periodat durch Natriumsulfit bei einer Endkonzentration von 20 mM und pH 5 für 10 min bei Raumtemperatur. </li><li> Einführung Hydrazid-derivatisierten Harzes in die Peptidlösung in einem Verhältnis von 1:4 (Harz-Lösung) zu koppeln Glycopeptide zu dem Harz. Inkubieren der Reaktion bei 37 ° C für 1-2 Tage mit End-zu-End-Rotation für eine vollständige Kopplung. </li><li> Entfernen der nicht gebundenen Peptide durch Waschen des Harzes zweimal mit 1 ml von jeder der folgenden: DI-Wasser, 1,5 M NaCl, Methanol und 80% Acetonitril. Schließlich 3x Waschen des Harzes mit 1 ml 100 mM NH 4 HCO 3 bei pH 8, um den Puffer des Systems austauschen zu 100 mM NH 4 HCO 3. <ol><li> Sammle den Überstand und die Waschschritte für die Analyse von ungebundenen Peptiden (optional). </li></ol></li><li> Lösen der N-Glycopeptide vom Harz durch PNGase F in einer Inkubation über Nacht bei 37 ° C mitN-Ende-zu-Ende-Rotation. Sammeln Sie die freigesetzten Peptide durch Zentrifugation und einer 80% Acetonitril waschen. Trocknen Sie die erhaltene Lösung in der Speedvac für LC-MS-Analyse. </li></ol> 4. Weitere Fraktionierung (Optional) Zur weiteren Vereinfachung Probenkomplexität, Fraktionierung der N-Glycopeptiden. Beispielsweise abgeblasen, und der getrockneten Peptide in 10 mM Ammoniak-Formiat, 20% Acetonitril pH 3 und mit starken Kationenaustauscher (SCX) gereinigt, um die Peptide zu fraktionieren. Trocknen des erhaltenen Elutionsmittel und dann analysiert die erhaltenen Peptidfraktionen durch Umkehrphasen-LC-MS 8,17,18. 5. Reinigung der frei N-Glycopeptide (Optional) Wenn Bedenken erheben für die mögliche Kontamination der Peptide, wieder aufzulösen die getrockneten Peptide in 0,1% Ameisensäure und verwenden Sie einen MCX SPE-Säule, um die Peptide weiter zu reinigen, bevor Umkehrphasen-LC-MS-Analyse. <strong&gt; Hinweis: Datenbanksuchparameter. Bei der selektiven Spaltung von N-Glycopeptide aus dem Harz, wandelt die PNGase F-N-Glycan verknüpft Asparagin zu einer Asparaginsäure. Daher besteht ein 0,9840 Da Massenverschiebung der befreiten N-Glycopeptide. Diese Peptide genau zu identifizieren, muss diese Änderung der Suchparameter mit gemeinsamen Abänderungen wie der Carbamidomethylierung des Cystein und Oxidation der Methionin zugesetzt werden.

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Die Autoren erklären, dass sie keine finanziellen Interessen konkurrieren.

Hier stellen wir ein Glycopeptid-Capture-Strategie zur Profilierung Zelloberflächenproteine. Das Verfahren kann angewendet werden, um sekretierte Proteine, wie sie in Blut, wie auch in anderen Körperflüssigkeiten oder Zellkulturmedien zu studieren. Der Erfolg des Verfahrens beruht auf der vollständigen Verdau von Proben; Daher ist ein SDS-PAGE-Charakterisierung der Effizienz der Verdauung notwendig, insbesondere für die erste Zeitanalyse einer Probe. Eine vollständige Verdauung kann ei...

Zelloberflächen-Proteine ​​interagieren mit der extrazellulären Umgebung und leiten Signale von der Außenseite zur Innenseite der Zelle. Somit sind diese Proteine, einschließlich extrazellulären Matrixproteinen, spielen wichtige Rollen in allen Aspekten der Zellbiologie und der Physiologie von Proliferation, Wachstum, Migration, Differenzierung Alterung und so weiter. Oberflächenproteine ​​funktionieren durch die Interaktion mit anderen Zellen, Proteinen und kleinen Molekülen 03.01. Molekulare Charakterisierung von Zelloberflächenproteinen ist von großem Interesse, nicht nur für Biologen, sondern auch für die pharmazeutische Industrie, da mehr als 60% der Medikamente, Zelloberflächenproteine ​​4 ausgerichtet. Tandem-Massenspektrometrie (MS), mit seiner überlegenen Sensitivität, Genauigkeit und Durchsatz für die Identifizierung von Proteinen und Peptiden, wurde ein leistungsfähiges Werkzeug für die globale proteomische Studien 5,6. Doch stellen Oberflächenproteine ​​erhebliche Herausforderungen zu MS-basierte Proteomik, wiedie meisten Oberflächenproteine ​​gibt es in geringen Mengen und mit schweren Veränderungen. Die Membran-überspannenden Regionen der Oberflächenproteine ​​machen sie hydrophob ist; Dies ist insbesondere der Fall bei Multitransmembranproteine. Es ist daher schwierig, Membranproteine ​​in wässrigen Lösungen ohne Zuhilfenahme eines Detergens zu lösen; Jedoch ist die Verwendung von Waschmitteln in der Regel unterdrückt die Leistung der HPLC und MS 1,7,8 Proteinidentifikation. Daher Membranproteine ​​wurden schlecht in direkten LC-MS basierte Proteomik gekennzeichnet. Die Glykosylierung ist eine der wichtigsten und reichlich post-translationale Modifikationen, die sich in Zelloberflächenproteine ​​9. Die enorme Komplexität und Heterogenität der Glykane behindern Peptide &#39;MS-Signal 10. Dennoch haben mehrere proteomische Methoden dieses einzigartige Modifikation verwendet werden, um Oberflächenproteine ​​zu bereichern und die Zuckerreste von Proteinen vor der LC-MS-Analyse zu entfernen. Diese Methodes sind Lektin-basierte Affinitätseinfang 11 und Hydrazid-basierten oder Borsäure Chemie Capture 12 sowie hydrophile Trennungen 8,13. Die Entfernung der Glykane verwandelt Membranproteine, regelmäßige Proteine ​​und vereinfacht die MS Charakterisierung drastisch. Da Glykosylierung in sezernierte Proteine, die eine hohe Löslichkeit im Gegensatz zu Membranproteine ​​erfolgt auch, viele glycoproteomische Methoden für lösliche Proteine ​​optimiert und tendenziell niedrigere Glycopeptid Selektivität und Empfindlichkeit, wenn sie eingesetzt werden, um Proteine ​​8,14 Membran haben. Andere Verfahren bestehen auch zu bereichern, insbesondere Zelloberflächenproteine, wie z. B. jene, die Ultrazentrifugation 15 und Markierungsstrategien 16. Ein detaillierter Vergleich zwischen unserem Verfahren und anderen bestehenden Methoden zur Charakterisierung von Membranproteinen durchgeführt wurde kürzlich 17, und die Ergebnisse zeigten, daß unser Verfahren ebenso gut ausführen kann, wenn nichtbesser, als alle gegen Membran Proteomics Methoden, aber mit höheren Einfachheit. Um Forscher mit dieser Methode, die wir hier ausführlich ein allgemeines Protokoll. Diese Methode integriert mehrere Vorteile der bestehenden Glycoproteomikstrategie Strategien und ist speziell für Membran-Glykoproteine ​​entwickelt, doch die Methode funktioniert genauso gut für sekretierte Proteine. Die Merkmale dieses Verfahrens sind: 1) eine vollständige Solubilisierung von Membranproteinen, 2) eine Anreicherung von Glycopeptiden anstelle von Glykoproteinen, die potentielle sterische Behinderung zu vermeiden, wenn unter Verwendung eines festen Erfassungs Substrat, 3) die Verwendung von Hydrazid-Chemie, um kovalente Bindungen zwischen Form Glycopeptide und die Erfassung Substrat, so dass das gebundene Glycopeptide können stringente Waschungen für Hoch glycoselectivity tolerieren, und 4) die Fähigkeit, die gesamte Aufnahmevorgang in einem Rohr zur Probenverlust reduziert und verkürzt die Dauer Verfahren durchzuführen. Nach Durchführung dieses Verfahrens, um das Studium eine variableety von biologischen Proben, einschließlich Zellen und Geweben beobachteten wir eine hohe Selektivität (&gt; 90%) an Glycoproteine ​​8,17,18.

<table border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DTT</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646563</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCEP</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodoacetamide</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A3221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rapigest SF</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186001860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium periodate</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">311448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNGase F</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:119px;">P0704S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Affi-Gel Hz Hydrazide gel</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:119px;">153-6047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:199px;">Worthington Biomedical</td>
			<td style="width:119px;">LS02115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sep-Pak C-18 cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">WAT054955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor coctail</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:199px;">Amersco</td>
			<td style="width:119px;">568</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium sulphite</td>
			<td style="width:199px;">Caledon</td>
			<td style="width:119px;">8360-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Invertase</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">I0408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alpha-1 trypsin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ribonulease B</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">R7884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Avidin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ovalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">C0755</td>
			<td></td>
		</tr>
	</tbody>
</table>

glycopeptide capture for cell surface proteomics

Zelloberflächenproteine, einschließlich extrazellulären Matrixproteinen, sich an allen wichtigen zellulären Prozessen und Funktionen, wie Wachstum, Differenzierung und Proliferation. Eine umfassende Charakterisierung dieser Proteine ​​bietet reichhaltige Informationen zur Entdeckung von Biomarkern, Zelltyp-Identifikation und Drogenzielauswahl, als auch helfen, unser Verständnis der Zellbiologie und Physiologie voraus. Oberflächenproteine ​​stellen jedoch erhebliche analytische Herausforderungen, die aufgrund ihrer inhärent niedrigen Fluss, hohe Hydrophobie und schwere post-translationale Modifikationen. Unter Ausnutzung der vorherrschenden Glykosylierung auf Oberflächenproteine, führen wir hier ein Hochdurchsatz-Glycopeptid-Capture-Ansatz, der die Vorteile von mehreren vorhandenen N-Glycoproteomikstrategie Mittel integriert. Unsere Methode kann die Glykopeptide von Oberflächenproteinen abgeleitet bereichern und entfernen ihre Glykane für einfache Proteomik mittels LC-MS. Die beschlossene N-glycoproteome umfasst die informationen von Protein Identität und Quantität sowie ihre Websites der Glykosylierung. Dieses Verfahren hat zu einer Reihe von Studien in Bereichen wie Krebs, Stammzellen und Arzneimitteltoxizität angewendet. Die Beschränkung des Verfahrens liegt in der geringen Häufigkeit von Oberflächenmembranproteinen, so dass eine relativ große Menge von Proben für die Analyse erforderlich im Vergleich zu Studien am cytosolischen Proteine ​​zentriert.

Ein repräsentatives Flussdiagramm des experimentellen Verfahrens ist in Fig. 1 zusammengefaßt. Die Etikettierung und weitere Fraktionierung Schritte sind optional und Details sind in einer Veröffentlichung 18 beschrieben. Eine weitere Möglichkeit ist die unveränderten Peptide, die nicht mit dem Harz reagieren zu analysieren. Die Vorteile des Analysierens der unmodifizierten Peptide umfassen die Identifizierung von potentiellen nicht-glykosylierte Peptide und Proteine, wie Claudinen in ti...

Watch this Scientific Journal Video about Glycopeptide Capture for Cell Surface Proteomics at JoVE.com

Glycopeptide Capture for Cell Surface Proteomics

Zelloberflächen-Proteine ​​sind biologisch wichtige und weithin glykosyliert. Wir stellen hier eine Glykopeptid-Capture-Ansatz für einfache LC-MS basierte Proteomanalysen löslich zu machen, zu bereichern, und deglycosylieren diese Proteine.

Glycopeptid-Capture für Zelloberflächen Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

glycopeptide-capture-for-cell-surface-proteomics

Research

JoVE Journal

Biology

11.2K Aufrufe.  Simon Fraser University.  Zelloberflächen-Proteine ​​sind biologisch wichtige und weithin glykosyliert. Wir stellen hier eine Glykopeptid-Capture-Ansatz für einfache LC-MS basierte Proteomanalysen löslich zu machen, zu bereichern, und deglycosylieren diese Proteine. 

Serie: Glycopeptide Capture for Cell Surface Proteomics

Cell Capture Using a Microfluidic Device

Zell-Capture mit einer mikrofluidischen Vorrichtung

Forschung

Biologie

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

High-Throughput-Kristallisation von Membranproteinen mit dem Lipidische Bicellen Methode

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidität-basierte extrazelluläre Wechselwirkung Screening (AVEXIS) für die Erkennung von Scalable Low-Affinität extrazellulären Rezeptor-Ligand-Wechselwirkungen

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabolische Markierung und Membran Fraktionierung für Vergleichende Proteomanalyse von<em&gt; Arabidopsis thaliana</em&gt; Fahrwerk Zellkulturen

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Proteomik Probenvorbereitung von Formalin fixiert und in Paraffin eingebetteten Gewebe

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Capture Compound Mass Spectrometry - Ein leistungsfähiges Tool zum Identifizieren Novel c-di-GMP Effektorproteinen

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Nicht-enzymatische, Serumfreie Zellkultur von Pre-invasive Brustläsionen für Spontane Erzeugung Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Selected Reaction Monitoring-Massenspektrometrie für Absolute Proteinquantifizierung

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

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 ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Oberfläche Verbreitung und Immunfärbung von Hefechromosomen

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...