Questo lavoro è stato sostenuto dal Technologies Proteomica clinica per il cancro iniziativa, 5U24CA126477-04.

1) Preparare lectina coniugato colonne POROS <ol><li> Indossare una maschera per la protezione contro l&#39;inalazione di Poros-AL perline durante le fasi di 1,1-1,2. Pesare la quantità desiderata di perline POROS (100 mg beads/300 microlitri di volume finale) e trasferire in una provetta pulita eppendorf. </li><li> Lavare le perline con l&#39;aggiunta di 1 ml di soluzione salina tampone fosfato (PBS). Perle di pellet per centrifugazione in una microcentrifuga alla massima velocità per 3 minuti. Rimuovere il surnatante e ripetere il lavaggio. </li><li> Pesare la quantità desiderata di lectina non coniugato (1 - 4 mg / 200 perline microlitri) e trasferirlo in una provetta pulita eppendorf. Aggiungi PBS per formare un 5 - 20 mg / ml soluzione. Riserva di 25 ml di questa soluzione. </li><li> Trasferire la soluzione rimanente lectina ai talloni POROS. Aggiungi cyanoborohydride di sodio ad una concentrazione finale di 50 mm. Posizionare il tubo su una sedia a dondolo e reagire notte a temperatura ambiente. (Nota:. Cyanoborohydride sodio è tossico e deve essere gestito in una cappa aspirante I rifiuti contaminati devono essere smaltite in modo appropriato.) </li><li> Pellet le perline POROS come al punto 1.2. Rimuovere il surnatante e salvarli come il post-coniugazione soluzione. </li><li> Lavare le perline con 1 mL di tampone tempra (1 M Tris-Cl, pH 7,4). Perline pellet come al punto 1.2 e in modo sicuro surnatante. </li><li> Bloccare i siti rimanenti reattivi sulle palle POROS con 1 mL di tampone tempra. Aggiungi cyanoborohydride di sodio ad una concentrazione finale di 50 mm. Posizionare il tubo su una sedia a dondolo e incubare a temperatura ambiente per 30 min. </li><li> Perline pellet come al punto 1.2 e scartare il surnatante. </li><li> Lavare con 1 ml di perline 1 M NaCl. Perline pellet surnatante e gettarlo. Ripetere quattro volte per un totale di cinque lavaggi. Le perle sono ora pronti per il confezionamento. </li><li> Per determinare la quantità di proteina che è stato coniugato con le perline POROS, eseguire un test di concentrazione di proteine ​​sulle soluzioni lectina pre-coniugato e post-coniugati. La differenza di concentrazione è la quantità di proteina che è stato coniugato con le perline. La quantità di proteina coniugata per volume cordone uguale la concentrazione di lectina sul perline. Concentrazioni di lectina target sono tra 2 -20 mg / ml. </li></ol> 2) Imballaggio della lectina coniugato perle POROS in una colonna PEEK <ol><li> Assemblare il sistema di imballaggio (segue descrizione) e il supporto su un supporto ad anello in metallo. Il sistema è composto da imballaggio, dal basso verso l&#39;alto, limitatore di pressione, colonna fine accoppiatore 1, fritta, colonna (2 x 50 mm), colonna accoppiatore end 2, connettore colonna, colonna fine accoppiatore 3, colonna (4,5 x 50 mm), fine accoppiatore colonna 4 e il montaggio finale. La colonna superiore funge da serbatoio per il materiale di imballaggio. </li><li> Risospendere le sfere POROS nel volume desiderato di tampone A (10 mM Tris, pH 7,4, 150 mM di cloruro di sodio, cloruro di calcio 10 mM, 10 mM cloruro di magnesio). Se una colonna di imballaggio (~ 200 microlitri di volume letto), risospendere in 400 A. Buffer microlitri </li><li> Trasferimento coniugato perline POROS nella colonna superiore (il serbatoio). Aggiungi tampone A al serbatoio fino a quando il buffer raggiunge la cima della colonna. Posizionare con delicatezza il raccordo finale sulla parte superiore della colonna, cercando di evitare bolle d&#39;aria nella colonna. Collegare il raccordo fine della colonna superiore per il sistema HPLC. </li><li> Pacco la colonna che scorre tampone A attraverso il sistema. Inizia con una portata di 0,5 ml / min. Aumentare la velocità di flusso di 0,5 mL / min ogni minuto fino a quando 4 mL / min o la pressione massima (3000 psi) è stato raggiunto. Continua alla portata massima possibile fino almeno 35 ml di tampone A sono passati attraverso la colonna. </li><li> Spegnere le pompe e permettere la pressione sulla colonna per scendere a &lt;20 psi. </li><li> Delicatamente, smontare il sistema di imballaggio, a partire dall&#39;alto. Quando la colonna 2 della fine accoppiatore è raggiunto, rimuovere con attenzione. Alcuni imballaggi sono da estrusione dalla parte superiore della colonna. Con una lametta o simili bordo affilato, rimuovere delicatamente le perline estrusione, lasciando una superficie delle sfere che è anche con la parte superiore della colonna. Non applicare la pressione ai talloni mentre fanno questo. </li><li> Sganciare la colonna confezionato dallo stand anello. Inserire una nuova fritta in un accoppiatore nuova fine e girare la colonna riempita oltre a collegare questo con la fritta / fine accoppiatore sul aperti (ex superiore) fine colonna. </li><li> Etichetta di colonna in modo appropriato. Ora è pronto per l&#39;uso. Quando non in uso, la colonna possono essere memorizzati in PBS con sodio azide 0,02% a 4 ° C per un massimo di 6 mesi. </li></ol> 3) Programmare il metodo HPLC <ol><li> I dettagli della programmazione del HPLC varierà a seconda delle specificità dei software del produttore. Usiamo un paradigma MG4 Michrom HPLC. Su questa macchina, i metodi sono costruiti e accedere sotto la scheda &quot;Impostazioni&quot; nella parte superiore dello schermo. Programmare il seguente metodo: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tempo </td><td> Portata (microlitri / min) </td><td> % A </td><td> % B </td></tr><tr><td> 00:00 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 09:00 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 09:01 </td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13:50 </td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13:51 </td><td> 3000 </td><td> 100 </td><td> 0 </td></tr><tr><td> 19:50 </td><td> 3000 </td><td> 100 </td><td> 0 </td></tr></tbody></table> Tampone A: 10 mM Tris, pH 7,4, 150 mM di cloruro di sodio, cloruro di calcio 10 mm, 10 mM di cloruro di magnesio Buffer B: 0,5 M acido acetico Il rilevatore di raggi UV deve essere programmato per leggere a 280 nm 0:00-19:50. L&#39;asse y deve essere regolato per rilevare a basso assorbimento livelli, ad esempio, 0 -. 50 UA. </li><li> Se un campionatore automatico è disponibile, programma il seguente calendario per la raccolta della frazione. In caso contrario, raccogliere le frazioni a mano, come indicato durante l&#39;esecuzione di cromatografia. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Frazione </td><td> Ritardo alla raccolta di iniezione del campione </td><td> Durata della raccolta (min) </td></tr><tr><td> Flow-through </td><td> 2,8 </td><td> 4,1 </td></tr><tr><td> Legato </td><td> 9 </td><td> 2,6 </td></tr></tbody></table></li></ol> 4) Preparare i campioni per HPLC <ol><li> Prima di utilizzare in questo protocollo, plasma umano dovrebbe essere impoverito del 14 proteine ​​più abbondanti, con una colonna MARS (Agilent, Santa Clara, CA). Impoverito plasma umano lattoferrina e invertasi lievito deve essere valutata individualmente tripsina-digerito e dissalati come descritto in precedenza 1. </li><li> Per preparare la lectina specifica lattoferrina standard, eseguire la cromatografia su 50 ug-tripsina digerito lattoferrina oltre sia il AAL o la colonna SNA usando il metodo nella parte 3. La frazione legata conterrà lectina specifica glicopeptidi lattoferrina. Neutralizzare i campioni come nella parte 4.4, e desalt come in parte 6. Risospendere il campione in 1 ml di tampone A. Il campione risultante è pronto per l&#39;uso. </li><li> Tre sperimentale replica verrà eseguita. Per la preparazione del campione per tre repliche, aggiungere 3 pmol di tripsina-digerito invertasi e 1 ml di lectina specifica lattoferrina (sia AAL-LAC o SNA-ALC) a 30 microlitri MARS-impoverito, tripsina-digerito, equivalenti al plasma (PE). Un PE è definito come il volume di plasma non-processed/intact/original da cui provengono una data quantità di trattati al plasma (MARS-tripsina impoverito e digerito). Aggiungi tampone A al campione di raggiungere un volume finale di 330 microlitri. </li><li> Aggiungi buffer di neutralizzazione (1 M Tris pH 8,0), per le provette del campione che raccoglierà eluato. Il numero e il volume di flaconcini di lavorare con dipenderà dalle costrizioni del campionatore automatico. Per il paradigma MG4, un flaconcino raccoglie la flow-through e due raccogliere l&#39;eluato. Richiederà circa 1,5 ml Tampone Tris di neutralizzare 1 ml di eluato. PH neutralizzato obiettivo è 7,0-8,0; test del pH prova finale con una cartina di tornasole. </li></ol> 5) Eseguire la cromatografia <ol><li> Tutte le piste vuote e il campione che seguono utilizzerà il metodo descritto nella parte 1. La colonna e buffer sono a temperatura ambiente durante l&#39;uso. Le colonne sono conservati a 4 ° C, mentre i buffer vengono conservati a temperatura ambiente. </li><li> Attaccare o il AAL o la colonna lectina SNA al sistema HPLC ed eseguire due metodi vuoto (iniezione di buffer solo A). Assicurarsi che la colonna lectina corrisponde alla lectina specifica lattoferrina (Parte 2). </li><li> Cromatografo i tre campioni di plasma, l&#39;iniezione di un vuoto tra ogni campione iniettato, per un totale di 7 corre HPLC. Frazioni da corre vuoto non devono essere raccolti. </li></ol> 6) Desalt raccolti frazioni Per ogni frazione utilizzare uno da 1 ml Acque Oasis HLB cartuccia SPE come segue: <ol><li> Fissare il numero di cartucce necessarie al collettore vuoto. </li><li> Bagnate ogni cartuccia con 1 mL ACN 80% in 1% di acido formico (vacuometro sul collettore dovrebbe leggere 5-20 lnHg). </li><li> Equilibrare cartucce con 1,5 ml di acido formico allo 0,1% (vacuometro sul collettore dovrebbe leggere 5-20 lnHg) </li><li> Carico intero volume di un campione su una cartuccia (Vuotometro sul collettore dovrebbe leggere 2 -. 2,5 lnHg, e portata non dovrebbe superare 1 ml / min) </li><li> Lavare le cartucce con 3 x 1 ml di acido formico allo 0,1% (Vuotometro sul collettore dovrebbe leggere 5-20 lnHg) </li><li> Peptidi eluire / glicopeptidi in tubi eppendorf etichettati con 1 x 1 mL acetonitrile 80% a 0,1% acido formico. (Vuotometro sul collettore dovrebbe leggere 2-2,5 lnHg, e portata non dovrebbe superare 1 ml / min.) Un tubo di raccolta unica dovrebbe essere utilizzato per ogni cartuccia. </li><li> Neutralizzare eluato con l&#39;aggiunta di 60 microlitri di bicarbonato di ammonio 0,5 M per ogni tubo di raccolta. Obiettivo neutrpH è alized è 7,0-8,0; pH prova finale con una cartina di tornasole. </li><li> Concentrato peptidi eluiti / glicopeptidi a ~ 50 microlitri eseguendoli su una centrifuga a vuoto (ad esempio, Thermo Savant SpeedVac) per circa 2 ore a 35 ° C. </li></ol> 7) PNGase F-digestione <ol><li> PH del campione di prova per assicurarsi che sia tra 7,0-8,0. Se necessario, aggiungere 50 bicarbonato di ammonio mM per aumentare il pH. Se il volume del campione finale è&gt; 100 l, campione speedvac fino a quando non è tra 50-100 microlitri. </li><li> Aggiungere 0,5 microlitri (250 U) glicerolo senza F PNGase a ciascuna provetta ed incubare a 37 ° C durante la notte. </li></ol> 8) campioni Suggerimento Zip <ol><li> A partire da PNGase F-digerito campioni, ziptip ognuno nel modo seguente. </li><li> Ziptip bagnato con 10 microlitri acetonitrile al 100%, seguita da 10 microlitri acetonitrile 80%, 0,1% di acido formico. </li><li> Equilibrare ziptip con 20 microlitri di acido formico 0,1%. </li><li> Caricare il campione attraverso la ziptip pipettando su e giù attraverso la matrice ziptip 10 volte. </li><li> Lavare la ziptip pipettando 10 microlitri di acido formico 0,1%, 5 volte. </li><li> Eluire il campione in una provetta eppendorf pulita con 10 microlitri acetonitrile 80%, 0,1% di acido formico. </li><li> Ripetere l&#39;eluizione e aggiungere il secondo 10 microlitri nel tubo stesso. </li><li> Speedvac i campioni ad un volume di &lt;2 microlitri. </li><li> Risospendere i campioni in 20 microlitri di acido formico 0,1%. I campioni sono ora pronti per l&#39;analisi LC-MS/MS. </li></ol> 9) Risultati Rappresentante: La coniugazione POROS rendimenti generalmente concentrazioni lectina sul tallone nel range di 2 - 20 mg / mL. Questo è ottimale per cromatografia di affinità. Cromatografia lectina di tripsina-digerito MARS-impoverito plasma umano arricchisce tipicamente glicopeptidi nella frazione legata. Mentre il glicopeptidi sono PNGase F-trattati prima LC-MS/MS, essi sono identificati come peptidi con la N-linked sequenza consenso, NXS / T (dove X è un qualsiasi residuo, prolina), in cui l&#39;asparagina è stato convertito in un acido aspartico attraverso l&#39;azione di PNGase F. Peptidi con queste caratteristiche sono considerati peptidi deglicosilata (o &quot;deglycopeptides&quot;). In media la flow-through (FT) frazione da AAL o SNA cromatografia contiene deglycopeptides 2-4%, mentre il 30-50% dei peptidi recuperato nella frazione legata sono deglycopeptides. Di solito, usando una Elite QStar, 1000-1300 peptidi saranno identificati nella frazione FT, e 200-400 peptidi saranno identificati nella frazione legata. Due o più deglycopeptides invertasi distinte devono essere osservate nella frazione FT e due o più distinte deglycopeptides lattoferrina nella frazione legata (Tabella 1). <img src="/files/ftp_upload/1398/1398table.jpg" alt="Tabella 1" /> Tabella 1.

<ol>
	<li>Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=multiplexed+assays+for+low+abundance+proteins+in+plasma+by+targeted+mass+spectrometry+and+stable+isotope+dilution.">multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.</a> Mol Cell Proteomics. 6, 2212-2229 (2007).</li></ol>

Questo protocollo fornisce un metodo rapido per isolare e identificare glicopeptidi in una glycan specifico modo. Come glicosilazione varia ampiamente all&#39;interno di un organismo, in particolare durante lo sviluppo e la patogenesi, questa tecnica può essere applicabile per affrontare una vasta gamma di domande. In particolare, stiamo usando il metodo per arricchire selettivamente glicopeptidi che sono stati modificati con associata al cancro epitopi come primo passo verso la scoperta di biomarcatori.

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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>Human plasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>We make this in house</td>
</tr>
<tr>
<td>Human lactoferrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L0520</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Yeast invertase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I0408</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Oass HLB SPE cartridges</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT094225</td>
<td>1 cc volume</td>
</tr>
<tr>
<td>ZipTip pipet tips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>ZTC1 8M0 96</td>
<td>Millipore, C18 for 10 ml pipette</td>
</tr>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP154-1</td>
<td>99%</td>
</tr>
<tr>
<td>Ammonium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A6141</td>
<td>99%</td>
</tr>
<tr>
<td>Calcium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4901</td>
<td>96%</td>
</tr>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>98%</td>
</tr>
<tr>
<td>Acetic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>242853</td>
<td>99.7%</td>
</tr>
<tr>
<td>pH indicator paper</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>M95903</td>
<td>Range 0-14</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A998</td>
<td>99.9%</td>
</tr>
<tr>
<td>Formic acid</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28905</td>
<td>99%</td>
</tr>
<tr>
<td>Phosphate-buffered saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-136</td>
<td>Without calcium and magnesium</td>
</tr>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>71289</td>
<td>99.5%</td>
</tr>
<tr>
<td>PNGase F</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>P0705L</td>
<td>Glycerol-free</td>
</tr>
<tr>
<td>Vacuum-filter flasks</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SCGVU05RE</td>
<td>0.2 mm pores</td>
</tr>
<tr>
<td>POROS-AL beads</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>1-6028-02</td>
<td>&nbsp;</td>
<tr>
<td>Aleuria aurantia lectin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1390</td>
<td>&nbsp;</td>
<tr>
<td>Sambucus nigra agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1300</td>
<td>&nbsp;</td>
<tr>
<td>Sodium cyanoborohydride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>296945</td>
<td>5.0 M solution</td>
</tr>		</tbody>
		</table>
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a lectin hplc method to enrich selectively-glycosylated peptides from complex biological samples

Glicani sono una classe importante di modificazioni post-traduzionali. Tipicamente presenti su secreto e molecole extracellulari, strutture glycan segnalare lo stato interno della cellula. Glicani sulle cellule tumorali tendono ad avere abbondante acido sialico e frazioni fucosio. Proponiamo che queste associata al cancro varianti glycan essere sfruttata per lo sviluppo di biomarcatori volti a diagnosticare in fase iniziale della malattia. Di conseguenza, abbiamo sviluppato una spettrometria di massa a base di flusso di lavoro che incorpora cromatografia su matrici di affinità formata da lectine, proteine ​​che si legano strutture glycan specifici. Le lectine Sambucus nigra (SNA) e Aleuria aurantia (AAL), che legano l&#39;acido sialico e fucosio, rispettivamente, sono state accoppiate in modo covalente a microsfere POROS (Applied Biosystems) e confezionata in colonne PEEK per cromatografia liquida ad alta pressione (HPLC). In breve, il plasma è stato impoverito dei quattordici proteine ​​più abbondanti con un sistema di rimozione più affinità (MARS-14; Agilent). Impoverito plasma è stato tripsina-digerito e separati in flow-through e le frazioni vincolato da SNA o AAL HPLC. Le frazioni sono stati trattati con PNGaseF per rimuovere N-glicani legate, e analizzati da LC-MS/MS su Elite QStar. I dati sono stati analizzati utilizzando il software Mascot. Il disegno sperimentale inclusi controlli positivi-fucosylated e sialylated umano glicopeptidi e lattoferrina negativo glicopeptidi mannosio controlli alto da Saccharomyces cerevisiae, che sono stati utilizzati per monitorare la specificità di cattura lectina. Le caratteristiche principali di questo flusso di lavoro includono la riproducibilità derivato dal formato HPLC, l&#39;identificazione positiva dei glicopeptidi catturato e PNGaseF trattati dai loro motivi Asn-Xxx-Ser/Thr deamidato, e valutazione della qualità utilizzando standard glicoproteina. Protocollo di ottimizzazione incluso anche determinare il rapporto appropriato di materiale di partenza alla capacità di colonna, individuando la cattura più efficiente e buffer di eluizione, e il monitoraggio della PNGaseF-trattamento per garantire la piena deglycosylation. Direzioni future includono l&#39;utilizzo di questo flusso di lavoro di effettuare la spettrometria di massa a base di esperimenti di scoperta sul plasma da pazienti affetti da tumore al seno e individui di controllo.

Watch this Scientific Journal Video about A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples at JoVE.com

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

Lectina coniugato perline POROS sono stati impiegati per HPLC. Standard glicopeptide serviti come controlli positivi e negativi. MARS-14 impoverito, tripsina-digerito plasmatiche umane è stato cromatografato e flow-through (FT) e le frazioni legato raccolti per analisi ESI-LC-MS/MS. Glicopeptidi sono stati arricchiti nella frazione legata, rispetto a FT.

Un metodo HPLC lectina per arricchire Peptidi selettivamente glicosilata da campioni biologici complessi

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

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15.9K Views.  University of California, San Francisco - UCSF. Lectina coniugato perline POROS sono stati impiegati per HPLC. Standard glicopeptide serviti come controlli positivi e negativi. MARS-14 impoverito, tripsina-digerito plasmatiche umane è stato cromatografato e flow-through (FT) e le frazioni legato raccolti per analisi ESI-LC-MS/MS. Glicopeptidi sono stati arricchiti nella frazione legata, rispetto a FT.

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

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T-wave mobilità ionica-spettrometria di massa: Basic procedure sperimentali per l&#39;analisi delle proteine ​​complesse

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

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Biologia

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Peptidi dalla libreria phage display modulare l&#39;espressione genica nelle cellule mesenchimali e potenziare Osteogenesi in difetti ossei Unicortical

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

A PCR-based metodo di genotipizzazione di distinguere tra le varietà selvatiche di tipo ornamentale e di Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

La logica, passaggi sperimentali, e le potenzialità di eterologa Biosintesi prodotto naturale Con l&#39;antibiotico Eritromicina Complesso A prodotti attraverso<em&gt; E. coli</em

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Un metodo per la microiniezione di<em&gt; Patiria minata</em&gt; zigoti

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

Purificare l&#39;Impure: sequenziamento metagenomi e Metatranscriptomes da campioni complessi animali associate

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Un ibrido metodo di estrazione del DNA per la valutazione delle comunità batteriche qualitativa e quantitativa di pollame campioni di produzione

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

Rimozione di materiali esogene dalla parte esterna della congelati core per Indagare sulla antica comunità biologiche ospitava Dentro

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

Preparazione e di osservazione di campioni biologici spessi da Scanning Transmission Electron Tomografia

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Metodo per l'etichettatura di trascrizioni in cellule individuali Escherichia coli per singola molecola di fluorescenza In Situ gli esperimenti di ibridazione