Ce travail a été soutenu par des subventions et des AG027818 AG030709 du NIH / NIA, 2005/2E de la Fondation Larry L. Hillblom, IIRG-07-58334 de l&#39;Association Alzheimer, et 07-65798 de la Californie ministère de la Santé publique.

1. La préparation de peptides <ol><li> Peser ~ 100-200 ug de peptide lyophilisé en utilisant une microbalance et transférer dans étiquetés, siliconé, microtubes de faible adsorbant. Ici, nous utilisons les séquences humaines de l&#39;Aß40, Aß42, la calcitonine, et GRF. </li><li> Ici, nous utilisons des peptides pré-traitées avec 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) pour obtenir homogène, sans agrégat préparatifs. Cette étape est nécessaire car pré-formé des agrégats induire l&#39;agrégation rapide des protéines amyloïdogénique, qui aboutissent à une mauvaise reproductibilité des expériences 5. D&#39;autres méthodes telles que la filtration et de la SEC peuvent également être utilisées pour obtenir sans agrégat préparatifs pour PICUP 6. </li><li> Pour traiter les peptides avec HFIP, pré-refroidir le conteneur de HFIP sur glace à l&#39;intérieur d&#39;une hotte porter une protection adéquate (HFIP est volatile et toxique). Refroidissement d&#39;une bouteille de 250 ml nécessite généralement 10-15 min. Ajouter HFIP de pré-réfrigérée tubes contenant lyophilisats peptide pour obtenir une concentration de peptide nominale de 0,5 mM. </li><li> Soniquer les solutions de peptide dans un sonicateur bain-marie pendant 5 min à température ambiante. </li><li> Vortex doucement et incuber les tubes pendant 30 min à température ambiante. </li><li> Réfrigérer les tubes sur la glace (pendant 1 min), et de diviser les solutions en 10-50-ul aliquotes de 0,6 ml, étiqueté faible adsorbant tubes. </li><li> Retirer HFIP par évaporation sous un léger courant d&#39;azote, ou laisser les tubes ouverts dans la hotte du jour au lendemain. Pour l&#39;évaporation pendant la nuit, placer les tubes ouverts dans un rack et les couvrir avec une grande feuille de Kimwipes pour empêcher la poussière et la contamination particulaire. </li><li> Exsiccate l&#39;HFIP restent sous vide dans un lyophilisateur, ou un concentrateur centrifuge pendant 30 min, ou dans un dessicateur attaché à une entrée vide pendant 2 h. Le produit final sera un film de peptide dans le bas des tubes de microcentrifugation. Si elle est correctement exsiccated, les tubes peuvent être stockés pendant de longues périodes étanches (mois) à -20 ou -80 ° C. </li></ol> 2. Solubilisation des peptides HFIP-traitée et la photo de réticulation <ol><li> Avant de solubiliser les peptides HFIP-traités pour la réticulation des réactions, on a besoin pour préparer la réticulation et des réactifs de trempe. </li><li> Peser le persulfate d&#39;ammonium (APS, M. 228,2 g / mol) et de préparer une solution à 20 mM dans phosphate de sodium 10 mM, pH 7,4. Mélanger à l&#39;aide d&#39;un vortex jusqu&#39;à ce que la solution est claire. </li><li> Préparer une solution mM de Tris (2,2-bipyridyle) dichlororuthénium (II) hexahydraté (RuBpy, M. 748,63 g / mol) dans 10 mM phosphate de sodium, pH 7,4. Mélanger à l&#39;aide d&#39;un vortex et de vérifier la dissolution complète. Protéger le tube de la lumière en utilisant du papier d&#39;aluminium. </li><li> Pour analyse SDS-PAGE après réticulation, un réactif trempe commode est de 5% β-mercaptoéthanol dans le tampon d&#39;échantillon 2 × SDS-PAGE. Alternativement, 1 M dithiothréitol (DTT, M. 154,5 g / mol) dans de l&#39;eau déminéralisée ou un tampon approprié peut être utilisé. </li><li> Films peptide HFIP-traitées sont dissous dans NaOH dilué d&#39;abord, puis un tampon phosphate de sodium est ajouté pour obtenir la concentration de peptide ~ 30 pM. Ajouter 60 mM de NaOH suivie d&#39;eau déminéralisée dans le tube contenant le film peptide tel que NaOH et l&#39;eau représentent 10 et 45% du volume final, respectivement. Racler le film peptide hors les murs à l&#39;intérieur du tube de centrifugeuse à l&#39;aide de la pointe, mélanger par pipetage de haut en bas, et soniquer pendant 5 min dans un sonicateur bain-marie. </li><li> Ajouter 45% phosphate de sodium 20 mM, pH 7,4, mélanger par pipetage, et centrifuger à 16000 g pendant 10 min. Mettez de côté des aliquotes de peptides non réticulés mélangé avec le réactif de trempe pour les contrôles négatifs. </li><li> Réglez la caméra obturateur de retard à 1 s. Au temps d&#39;irradiation plus élevés, de vastes réactions radicalaires peuvent provoquer la dégradation des protéines. Chargez l&#39;obturateur. Temps d&#39;irradiation peut avoir besoin d&#39;être optimisé en utilisant la méthode avec une nouvelle protéine. </li><li> Une réaction typique est PICUP effectuée dans un volume de 20 ul de réaction. Transfert 18 ul de la solution de peptide dans une paroi mince, claire, 0,2 ml, tube de PCR. </li><li> Pour la solution de peptide, ajoutez 1 RuBpy ul suivie par une APS ul et mélanger les réactifs par pipettage (la concentration finale de RuBpy et APS dans le mélange réactionnel sera de 0,05 et 1 mM, respectivement). </li><li> Placer rapidement le tube de réaction dans un flacon en verre de 1,8 ml. Placer le flacon à l&#39;intérieur du soufflet attaché à l&#39;avant du boîtier de l&#39;appareil. Fixez le protecteur d&#39;objectif et appuyez sur le déclencheur afin que l&#39;échantillon est irradié pendant 1 s à l&#39;intérieur du soufflet. </li><li> Après irradiation des échantillons, prendre rapidement le flacon hors du soufflet et le tube de PCR de la fiole. Vite arrêter la réaction en ajoutant 1 TNT ul ou 10 ul de tampon réduisant l&#39;échantillon PAGE. Répétez la réaction de chaque aliquote de peptide. </li><li> Les mélanges réactionnels peuvent maintenant être congelés à -20 ° C pour le stockage pour une durée maximale de 7 jours ou conservés sur la glace avant l&#39;analyse par SDS-PAGE et coloration argentée. </li></ol> 3. SDS-PAGE et coloration ruban-des produits de peptide réticulé <ol><li> Routine SDS-PAGE et coloration argentée sont effectuées pour visualiser réticulé peptides. </li><li> Charge 5 ul du mélange réactionnel par voie d&#39;obtenir ~ 130 pmol de peptide par voie. </li><li> Inclure des quantités similaires de non réticulé peptides pour la comparaison. Inclure également une échelle de protéines standards de rapprochement visuel de poids moléculaire des bandes peptidiques. </li><li> Exécutez le gel en utilisant un appareil d&#39;électrophorèse sur gel de la norme. Nous utilisons le XCell SureLock mini-cellule du système d&#39;Invitrogen et effectuer argent coloration en fonction de la publication Invitrogen IM-1002, Novex Pre-Cast Guide électrophorèse sur gel. </li></ol> Partie 4: Résultats Représentant (figure 1) <img src="/files/ftp_upload/old/1071_2009_1_19_13/Result no demarcation.png" alt="Figure 1" /> Figure 1: gels de polyacrylamide coloré à l&#39;argent montrant non réticulé (-) et photo-réticulé (+) GRF, la calcitonine, Aß40 et Aß42 SDS-PAGE et coloration argentée analyses de PICUP généré Aß40 oligomères montrent intensités des bandes à peu près similaire de monomère travers tétramère, suivie par une forte diminution de l&#39;abondance des oligomères supérieurs 7. Non réticulé Aß40 migre avec un compatible avec celui du monomère 7 M.. Aß42 montre une distribution oligomère distinctes taille. Aß42 oligomères comprennent 2-3 groupes 8. Le premier groupe, monomère par trimère, affiche intensité décroissante avec l&#39;ordre oligomère croissante. Dans le second groupe, une distribution gaussienne-like est observée entre tétramère et heptamère, avec un maximum à pentamère et hexamère. Le troisième groupe, qui n&#39;est pas représenté ici, contient des oligomères de M. ~ Da 30,000-60,000 8. Ces oligomères supérieurs de M. sont généralement observés à l&#39;aide SEC isolé LMW Aß42 mais pas peptide préparé par filtration ou 6 traitements HFIP. Non réticulé Aß42 produit principalement deux bandes, une bande de monomère et un large trimère / tétramère de bande, qui est un artefact induit par SDS 5,9. La calcitonine donne une distribution de taille oligomère qui diverge fortement d&#39;une diminution exponentielle monotone dans le monomère région par tétramère, suggérant la pré-existence de dimères, trimères, tétramères et 7. Le contrôle polypeptide, GRF, produit une distribution d&#39;oligomères monotone allant de dimères grâce hexamère tels que les montants relative apparente des oligomères baisse de façon exponentielle avec la masse moléculaire augmente. Dans d&#39;autres expériences, des oligomères supérieurs pourraient également être visualisés 7. Le nombre exact et les intensités relatives des bandes oligomère peut varier quelque peu entre les expériences en fonction de la concentration en protéines, la quantité de protéines chargées sur le gel et le temps utilisé pour l&#39;étape de développement dans le protocole de coloration argentée.

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</li><li>Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+(A&#946;)+assembly:+A&#946;40+and+A&#946;42+oligomerize+through+distinct+pathways.">Amyloid &#946;-protein (A&#946;) assembly: A&#946;40 and A&#946;42 oligomerize through distinct pathways.</a> Proc. Natl. Acad. Sci. USA. 100, 330-335 (2003).</li><li>Hepler, R. W., Grimm, K. M., Nahas, D. 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B., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Peptide+oligomerization+state+using+rapid+photochemical+crosslinking.">Determination of Peptide oligomerization state using rapid photochemical crosslinking.</a> Methods Mol. Biol. 299, 11-18 (2005).</li></ol>

PICUP a été développé à l&#39;origine pour étudier les complexes protéiques stables 2. La méthode a été appliquée plus tard à l&#39;étude quantitative de la protéine amyloïde métastable assemblées, y compris Aßi 10, prions et les maladies associées à la PrP Sc 11, et α-synucléine 12. Les facteurs les plus importants qui doivent être considérés lors de la conception d&#39;une expérience PICUP sont la stoechiométrie des réactifs, temps d&#39;irradiation, et la procédure ...

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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>Dolan-Jenner 200-W incandescent lamp</td>
<td>Other</td>
<td>Dolan-Jenner Industries</td>
<td>Model 170-D</td>
<td>The heat generated by the lamp does not affect samples for short incubation periods.</td>
</tr>
<tr>
<td>35-mm SLR camera body</td>
<td>Tool</td>
<td>Pentax</td>
<td>SP500 model</td>
<td>In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.</td>
</tr>
<tr>
<td>Clear, thin walled PCR tubes</td>
<td>Other</td>
<td>Eppendorf</td>
<td>951010006 supplied by Fisher L22-003-24</td>
<td>&nbsp;</td>
<tr>
<td>Glass vials (1.8 mL)</td>
<td>Other</td>
<td>Kimble Chromatography</td>
<td>60940A 2, supplied by Fisher 03-340-60</td>
<td>&nbsp;</td>
<tr>
<td>GRF</td>
<td>Reagent</td>
<td>Bachem</td>
<td>H-3695</td>
<td>&nbsp;</td>
<tr>
<td>HFIP</td>
<td>Reagent</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>A&beta;40 and A&beta;42</td>
<td>Reagent</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calcitonin</td>
<td>Reagent</td>
<td>American Peptide</td>
<td>22-1-10</td>
<td>&nbsp;</td>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. </td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7154-25 ML</td>
<td>Can be used when SDS-PAGE analysis is performed.</td>
</tr>
<tr>
<td>Novex Tricine SDS Sample Buffer (2x)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10-20%)</td>
<td>Other</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10x )</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Silver Express Staining Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6100</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides

L&#39;assemblage des protéines amyloïdogéniques en oligomères toxiques est un événement majeur dans la pathogenèse des maladies repliement des protéines, y compris la maladie d&#39;Alzheimer, de Parkinson et les maladies de Huntington, la sclérose latérale amyotrophique héréditaire, et diabète de type 2. En raison de la nature métastable de ces assemblages de protéines, il est difficile d&#39;évaluer leur distribution en taille oligomère quantitativement en utilisant les méthodes classiques, telles que l&#39;électrophorèse, la chromatographie diffusion de la lumière, la fluorescence, ou dynamique. Les oligomères de protéines amyloïdogéniques existent sous forme de mélanges métastable, dans lequel les oligomères se dissocient en monomères et d&#39;associer en grandes assemblées simultanément. PICUP stabilise les populations oligomère par reticulation covalente et lorsqu&#39;il est combiné avec des méthodes de fractionnement, tels que le sulfate de sodium électrophorèse sur gel de polyacrylamide dodecyl (SDS-PAGE) ou chromatographie d&#39;exclusion stérique (SEC), offre des instantanés de PICUP les distributions de taille oligomère qui existaient avant la Croix -lier. Ainsi, PICUP permet la visualisation et l&#39;analyse quantitative des populations en protéines métastable et peut être utilisé pour surveiller les relations d&#39;assemblage et décrypter entre les modifications de séquence et d&#39;oligomérisation<sup&gt; 1</sup&gt;. Mécaniste, PICUP implique la photo-oxydation de Ru<sup&gt; 2 +</sup&gt; Dans un tris (bipyridyle) Ru (II) complexes (RuBpy) à Ru<sup&gt; 3 +</sup&gt; Par irradiation avec une lumière visible en présence d&#39;un accepteur d&#39;électrons. Ru<sup&gt; 3 +</sup&gt; Est un puissant oxydant à un électron capable d&#39;abstraire un électron d&#39;une molécule de protéine voisins, générant une protéine radicale<sup&gt; 1,2</sup&gt;. Les radicaux sont instables et hautement réactives et donc des espèces disparaissent rapidement à travers une variété de réactions intra-et intermoléculaires. Un radical peut utiliser l&#39;énergie élevés d&#39;un électron non apparié à réagir avec un autre monomère de protéine formant un dimère, radical qui perd ensuite un atome d&#39;hydrogène et de formes stables, de manière covalente liés dimère. Le dimère peut alors réagir davantage à travers un mécanisme similaire avec des monomères ou des dimères d&#39;autres pour former des oligomères d&#39;ordre supérieur. Avantages de relative PICUP d&#39;autres photo-chimiques ou de méthodes de réticulation<sup&gt; 3,4</sup&gt; Inclure court (≤ 1 s) d&#39;exposition à la non-destructive de la lumière visible, pas besoin de<i&gt; Pré facto</i&gt; Modification de la séquence native, et de longueur nulle reticulation covalente. En outre, PICUP permet de réticulation des protéines au sein de pH et de température, y compris les paramètres physiologiques. Ici, nous montrons l&#39;application de PICUP de réticulation de trois protéines amyloïdogéniques les 40 - et 42-amyloïde β-résidus de protéines variantes (Aß40 et Aß42), et la calcitonine, et une protéine de contrôle, l&#39;hormone de croissance Facteur (GRF).

Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induit de réticulation des protéines non modifiées (PICUP) permet de caractériser la distribution de taille oligomère de mélanges de protéines métastable. Nous démontrons l&#39;application de PICUP à trois peptides représentatifs amyloïdogéniques les 40 - et 42-résidus formes d&#39;amyloïde β-protéine, et la calcitonine, et un peptide contrôle l&#39;hormone de croissance facteur de libération.

Photo-induite réticulation des protéines non modifiées (PICUP) appliqué à des peptides amyloïdogéniques

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

photo-induced-cross-linking-unmodified-proteins-picup-applied-to

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JoVE Journal

Biology

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

21.2K vues.  University of California, Los Angeles. Photo-induit de réticulation des protéines non modifiées (PICUP) permet de caractériser la distribution de taille oligomère de mélanges de protéines métastable. Nous démontrons l'application de PICUP à trois peptides représentatifs amyloïdogéniques les 40 - et 42-résidus formes d'amyloïde β-protéine, et la calcitonine, et un peptide contrôle l'hormone de croissance facteur de libération.

Vidéo: Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Séparation électrophorétique de protéines

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

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Biologie

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Coloration protéines dans les gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Une méthode CLHP lectine pour enrichir sélectivement peptides glycosylés d&#39;échantillons biologiques complexes

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolement du labiles complexes multi-protéiques par des In vivo Contrôlée Cellulaire réticulation et chromatographie d&#39;affinité immuno-magnétiques

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Microdissection laser appliquée au profil d&#39;expression génique de la Sous-ensemble de cellules provenant de la Drosophile Wing disque

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening Expression quantitative et purification appliquée à recombinant disulfure riches protéines venin produit dans<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Purification non chromatographique de polypeptides recombinants élastine et de leurs fusions avec des peptides et des protéines<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

L&#39;utilisation de MALDI-TOF spectrométrie de masse et une base de données personnalisée pour caractériser les bactéries indigènes à un environnement unique Cave (Kartchner, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Une méthode de sélection aptamères Structure commutation Appliqué à un colorimétrique Or nanoparticules Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

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.

Enrichissement de matrice extracellulaire de protéines à partir de tissus et la digestion en peptides pour l&#39;analyse de spectrométrie de masse

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