We thank Alberto Reinders for his work in optimizing the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredifor the annotation of the P. aeruginosa proteins. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

1. Preparazione del lisato <ol><li> Grow P. cellule aeruginosa in LB alla OD desiderata. NOTA: Per la guida: usare ≈ 100 ml cultura / campione per le culture di fase stazionaria e ≈ 500 ml cultur / campione per le culture di fase log (OD 600nm = 0.5). </li><li> Pellet mediante centrifugazione per 20 min a 5000 x g. </li><li> Risospendere 0.5-1 g di pellet in 1 ml di tampone di lisi (MES 6,7 mm, 6,7 mM Hepes, NaCl 200 mm, 6,7 mM Kac, DDT 1 mM, pH 7,5) e aggiungere inibitore della proteasi (completo mini, senza EDTA) e come DNaseI. </li><li> Lyse le cellule 3 passaggi attraverso una cella di pressione francese, a 20.000 psi (vedi Materiali List). </li><li> Ultra-centrifuga il lisato cellulare a 100.000 xg per 1 ora a 4 ° C. </li><li> Salvare il surnatante (passare al punto 2). </li><li> Lavare il pellet con 1 ml di tampone di lisi 1x pipettando su e giù. </li><li> Ultracentrifuga a 100.000 x g per 1 ora a 4 ° C. </li><li> Flash-congelare il pellet inazoto liquido e conservare a -20 ° C fino utilizzato per la cattura di proteine ​​di membrana (vedi punto 3). </li></ol> 2. Rimozione dei nucleotidi liberi c-di-GMP e altri (frazione solubile Only) <ol><li> Lavare una colonna PD10 dissalazione (vedi Materiali List) con 10 ml di tampone di lisi freddo. </li><li> Versare il supernatante (≈ 1 ml) sul PD10 per rimuovere nucleotidi. </li><li> Eluire con 4 ml di tampone di lisi freddo (500 gradini microlitri). </li><li> Selezionare le frazioni più concentrati, come determinato dal saggio Bradford, e piscina. </li></ol> (Solo Frazione Membrane) 3. Pellet Resuspension e solubilizzazione <ol><li> Risospendere il pellet in 500 a 1.000 ml di buffer di acquisizione 1x (senza detersivo) (vedi tabella 1). NOTA: Il pellet è difficile da riportare in sospensione. Si consiglia di prima pipetta su e giù con una pipetta per risospendere il pellet circa, poi utilizzare una siringa 27 G per omogeneizzare bene la soluzione. </li><li> Aggiungere 1% (w / v) di n-dodecil-β-D-maltopyranoside (DDM). </li><li> Incubare a 4 ° C per almeno 2 ore (o O / N) su una ruota girevole. </li><li> Ultracentrifuga a 100.000 x g per 1 ora a 4 ° C. </li><li> Raccogliere il surnatante. </li></ol> 4. Protein misura della concentrazione <ol><li> Misurare la concentrazione di proteine ​​mediante saggio Bradford (mediante saggio BCA per la frazione di membrana). </li><li> Impostare la concentrazione totale di proteine ​​a 10 mg / ml. </li></ol> 5. Capture <ol><li> Miscelare 300 mg di proteina con 1 mM di PIL, GTP, ATP, CTP, e 20 microlitri di tampone 5x cattura (HEPES 100 mM, 250 mM KAC, 50 MgAc mM, 5% glicerolo, pH 7,5) (Tabella 1). Regolare il volume di reazione di 100 microlitri con H 2 O. NOTA: il volume totale comprende il volume del CDG-CC e c-di-GMP nel caso della concorrenza controllo (vedere fase 5.3 e tabella 2). NOTA: tutti gli esperimenti sono stati eseguiti in 200 &amp;# 181; l strisce PCR 12-tubo (Thermo Scientific). NOTA: per la frazione di membrana, assicurarsi di mantenere sempre la concentrazione DDM sopra della concentrazione micellare critica (0,01% w / v) fino alla fase solubilizzazione delle proteine ​​in 8 M Urea (punto 7.1). </li><li> Incubare a 4 ° C per 30 minuti su una ruota girevole. </li><li> Aggiungere 10 mM CDG-CC (concentrazione finale). NOTA: includere un controllo senza CDG-CC (indicato come &quot;controllo tallone&quot;), e un controllo integrato con 1 mM c-di-GMP (&quot;controllo della concorrenza&quot;) (vedi tabella 2). NOTA: la concentrazione di CDG-CC può essere regolato da 1 a 10 micron. </li><li> Incubare a 4 ° C per almeno 2 ore (/ N per la frazione di membrana O) su una ruota girevole, al buio. </li><li> Dopo un breve giro, cross-link per l&#39;attivazione della porzione reattiva della CDG-CC con la luce UV per 4 minuti, utilizzando un CaproBox (vedi lista materiali, λ = 310 nm, Irradiance ≥10 mW / cm², distanza dalla sorgente = 2 centimetri). NOTA: rimuovere il coperchio delle strisce prima cross-linking. </li><li> Aggiungere 25 ml 5x tampone di lavaggio (5 M NaCl, 250 mM Tris, pH 7.5) e 50 ml di ben risospesa sfere magnetiche streptavidina, omogenizzare delicatamente. </li><li> Incubare a 4 ° C per 1 ora su una ruota girevole. </li></ol> 6. lavaggio Steps NOTA: (Magnet: vedi Materiali List). Iniziare con una cattura delle sfere magnetiche nel coperchio strip PCR, con il magnete. Quindi sostituire la striscia PCR con uno nuovo contenente la soluzione di lavaggio successivo. Rimuovere il magnete e risospendere le sfere, e incubare 2 min. Spin giù e sostituire il coperchio da un coperchio fresco. <ol><li> Fasi di lavaggio (frazione solubile solo) <ol><li> Lavare 6 volte in 200 microlitri tampone di lavaggio 1x. </li><li> Lavare una volta in 200 ml HPLC H 2 O. </li><li> Lavare 6 volte in 200 ml 80% acetonitrile. </li><li> Lavare 2 volte in 200 microlitri HPLC H 2 O. </li></ol></li><li> Fasi di lavaggio (membraFrazione ne solo) <ol><li> Lavare 5 volte in 200 microlitri 1x tampone di lavaggio + 0,1% DDM. </li><li> Lavare 2 volte in 200 microlitri 1x tampone di lavaggio + 0,05% DDM. </li><li> Lavare una volta in 200 microlitri 1x tampone di lavaggio + 0,025% DDM. </li><li> Lavare una volta in 200 microlitri 1x tampone di lavaggio + 0,0125% DDM. </li><li> Lavare 3 volte in 200 ml di ABC 100 mm (di bicarbonato di ammonio, NH 4 CO 3) + 2 M Urea. </li></ol></li></ol> 7. MS Preparazione del campione <ol><li> Risospendere le perline (direttamente nel coperchio) in 20 microlitri di ABC 100 mm (ABC 100 mm + 8 M Urea per la frazione di membrana) e trasferire in provette da 1,5 ml. </li><li> Frazione di membrana solo: incubare a 60 ° C per 5 min, agitando a 500 rpm. </li><li> Aggiungere 0,5 microlitri TCEP 200 mm (tris (2-carbossietil) fosfina) e incubare a 60 ° C per 1 ora, agitando a 500 rpm. Raffreddare a 25 ° C. </li><li> Aggiungere 0,5 ml di preparati al momento 400 mm iodoacetamide e incubare a 25 ° C per 30 minuti, agitando unT 500 rpm e al buio. </li><li> Aggiungere 0,5 ml di 0,5 M N-acetil cisteina e incubare a 25 ° C per 10 min agitando a 500 rpm. </li><li> Soltanto frazione membrana: aggiungere 1 ml Lys-C e incubare a 37 ° C, O / N. </li><li> Aggiungere 2 mg tripsina e incubare O / N a 37 ° C, agitando a 500 rpm (involucro in Parafilm per evitare l&#39;essiccazione). NOTA: i campioni possono essere conservati a -20 ° C in questa fase. NOTA: frazione di membrana solo: aggiungi ABC 100 mM per regolare la concentrazione di urea per &lt;2 M prima aggiunta di tripsina. </li><li> Brevemente centrifugare le provette e raccogliere perle con il magnete. </li><li> Trasferire il surnatante in un nuovo tubo di 1,5 ml (ripetere questo passaggio se perle sono lasciati). </li><li> Aggiungere 5 microlitri 5% TFA (acido trifluoroacetico) + 1 ml 2 M HCl (15 microlitri 5% TFA + 5 microlitri 2 M HCl per la frazione di membrana). </li><li> Colonne Condizione C18 MicroSpin (Il Gruppo Nest, MA, USA) con 150 microlitri acetonitrile (spin 20 sec a 2.400 rpm). </li><li> EquiLibrate le colonne C18 2 volte con 150 microlitri 0,1% TFA (spin 20 sec, 2.400 rpm). </li><li> Caricare il campione e spin 2 min, 2.000 giri. </li><li> Ricaricare il flow-through nella colonna e ripetere la fase di centrifuga (2 min, 2.000 rpm). </li><li> Lavare 3 volte con 150 microlitri 0,1% TFA, 5% acetonitrile (20 sec, 2.400 rpm). </li><li> Prendere una nuova provetta e eluire due volte con 150 microlitri 0,1% TFA, 50% acetonitrile (2 min, 2.000 rpm). </li><li> Asciugare i peptidi in una velocità-vac. </li><li> Risospendere in 40 microlitri 98% H 2 O, 2% acetonitrile, 0,15% di acido formico. </li><li> Sonicare 20 sec (ciclo impulso 0.5, l&#39;ampiezza del 100%, vedi Lista dei materiali) e spin down 5 sec, 12.000 rpm (da banco centrifuga). Vortex 10 sec, e spin down 5 sec, 12.000 rpm. Trasferire in un flaconcino HPLC per l&#39;analisi LC-MS / MS. </li><li> Congelare a -20 ° C. NOTA: i campioni possono essere conservati a -20 ° C in questa fase. </li></ol> 8. LC-MS / MS Analysis <ol><li> Eseguire un nano-LC (sistemi nano-LC) equipped con una colonna RP-HPLC (75 um x 37 cm) imballato con resina C18 (Magic C18 AQ 3 micron) usando un gradiente lineare dal 95% di solvente A (acido formico al 0,15%, 2% acetonitrile) e 5% di solvente B ( 98% acetonitrile, 0,15% di acido formico) al 35% di solvente B oltre 60 minuti ad una portata di 0,2 ml / min. </li><li> Analizzare i peptidi con LC-MS / MS (doppia pressione LTQ-Orbitrap Velos spettrometro di massa, collegato ad una sorgente ionica electrospray). NOTA: La modalità di acquisizione dei dati è stato impostato per ottenere una alta risoluzione MS scansione nella parte FT dello spettrometro di massa ad una risoluzione di 60.000 FWHM seguita da scansioni MS / MS nella trappola ionica lineare delle 20 ioni più intensi. Per aumentare l&#39;efficienza dei tentativi MS / MS, la carica dello stato di screening modus è stata attivata per escludere non assegnato e oneri singolarmente ioni. Collusione dissociazione indotta è stata innescata quando il precursore superato 100 conteggi ioni. La durata esclusione dinamico è stato impostato a 30 sec. Il tempo di accumulo di ioni è stato fissato a 300 msec (MS) e 50msec (MS / MS). </li></ol> 9. Database Search <ol><li> Scarica il P. aeruginosa NCBI database tramite la homepage NCBI ( <a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a> ). </li><li> Convertire gli spettri MS raw in file generici mascotte (MGF) utilizzando lo strumento di conversione MassMatrix ( <a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.py</a> ). </li><li> Cerca questo file mgf utilizzando MASCOTTE versione 2.3 contro il P. aeruginosa NCBI database contenente in avanti e le voci di proteine ​​reverse-esca. </li><li> Eseguire un in silico tripsina digestione dopo lisina ed arginina (meno seguito da prolina) tollerando due spaccature nella peptidi completamente triptici perse. </li><li> Impostare i parametri di ricerca database per consentire metionine ossidato (15,99491 Da) come modifiche variabile e carboxyamidomethylation (57,021464 Da) di residui di cisteina come modifica fisso. Per mascotte ci scrutaing scansioni ad alta risoluzione, impostare la tolleranza di massa precursore di 15 ppm e impostare la tolleranza di massa frammento di 0.6 Da. Infine, impostare la proteina FDR al 1%. </li><li> Importare le ricerche mascotte di P. Esperimenti aeruginosa CCMS in Scaffold (Proteomesoftware, versione 3), impostare i parametri per ottenere una proteina FDR vicino a 1%, ed estrarre il totale conta spettrali. </li><li> Per i risultati rappresentativi presentati in questo documento, abbiamo usato un test T accoppiato per confrontare l&#39;esperimento con il controllo della concorrenza, e solo considerato colpi con un valore di p inferiore a 0,1, e un rapporto di conteggio spettrale sopra 2 (sperimentare conteggi spettrali / controllo della concorrenza spettrale conta). </li><li> I dati possono essere esportati in qualsiasi software di foglio di calcolo per ulteriori analisi. </li></ol> 10. La quantificazione senza etichetta <ol><li> Importare i file raw in software progenesi LC-MS (Nonlinear Dynamics, versione 4.0). </li><li> Eseguire l&#39;allineamento LC-MS e la rilevazione in funzione Setti predefinitongs. </li><li> Esportare i dati in formato mgf da progenesi LC-MS. </li><li> Cerca gli spettri MS / MS con la mascotte contro il NCBI P. banca dati contenente aeruginosa in avanti e le voci di proteine ​​reverse-esca. </li><li> Importare i risultati di ricerca di database in progenesi LC-MS e mappare i peptidi identificazioni alle funzioni MS1. </li><li> Per la valutazione dei dati (calcolo dei livelli di significatività, rapporti fold-change) è stato utilizzato lo script ProteinSQAnalysis di SafeQuant (soglia: valore q inferiore a 0,1, il rapporto di conteggio spettrale sopra 2). </li></ol>

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The authors have nothing to disclose.

Particolare attenzione dovrebbe essere presa in diverse fasi del protocollo. La concentrazione proteica è un parametro critico con una concentrazione di 10 mg / ml di essere difficile da raggiungere quando le cellule vengono coltivate in condizioni di crescita specifici (ad esempio biofilm o piccole varianti colonia). Così, la risospensione pellet deve essere eseguita in un basso volume di tampone di lisi. Le concentrazioni di proteine ​​possono essere diminuiti di 8 mg / ml. Rispetto al metodo pubblicato...

c-di-GMP è una chiave secondo messaggero utilizzato dalla maggior parte dei batteri per controllare vari aspetti della loro crescita e il comportamento. Per esempio, c-di-GMP regola la progressione del ciclo cellulare, la motilità e l&#39;espressione di esopolisaccaridi e adesine superficiali 2-4. Attraverso il coordinamento di tali processi c-di-GMP promuove la formazione di biofilm, un processo che è associata con infezioni croniche di una gamma di batteri patogeni 5. c-di-GMP è sintetizzato da enzimi chiamati ciclasi diguanylate (DGCS) che ospitano un dominio catalitico GGDEF 4. Alcuni DGCS possiedono un sito inibitorio che regola le attività ciclasi su c-di-GMP vincolante. Il degrado di c-di-GMP è catalizzata da due classi distinte di fosfodiesterasi (PDE) che ospitano sia un EAL catalitico o HD-GYP dominio 6,7. La maggior parte delle proteine ​​effettrici noti che legano direttamente c-di-GMP appartengono a una delle tre classi di proteine: cataliticoGGDEF o EAL domini alleato inattivi e domini Pilz, piccoli interruttori molecolari che subiscono cambiamenti conformazionali su c-di-GMP vincolante 8. DGCS, PDE e Pilz proteine ​​sono ben caratterizzati e loro domini possono essere previsti in silico relativamente sicuro. Un particolare interesse è ora focalizzata sulla identificazione di nuove classi di effettori c-di-GMP. Alcuni effettori c-di-GMP con diversi motivi di legame sono stati descritti recentemente come la CRP / FNR Bcam1349 famiglia di proteine ​​in Burkholderia cenocepacia o FleQ regolatore trascrizionale in P. aeruginosa 9,10. Inoltre, riboswitches GMP-specifici c-di-stati recentemente identificati e indicati per controllare l&#39;espressione genica in modo c-di-GMP-dipendente 11. I motivi c-di-GMP vincolanti di diversi effettori solo sono mal conservati fare previsioni bioinformatici di tali proteine ​​difficile. Per risolvere questo problema, abbiamo sviluppato un metodo biochimico, che si basa sull&#39;utilizzo di una spe c-di-GMPfica Compound Capture combinata con spettrometria di massa 1,12,13. Abbiamo recentemente abbiamo progettato un nuovo trivalente c-di-GMP Compound Capture (CDG-CC, Figura 1) 1. Questo scaffold chimica è composto da: 1) un residuo c-di-GMP utilizzata come esca per catturare proteine ​​leganti c-di-GMP, 2) un gruppo reattivo UV-fotoattivabile utilizzato per attraversare link CDG-CC alle proteine ​​legate e 3) un biotina per isolare le proteine ​​catturate utilizzando sfere magnetiche rivestite di streptavidina. CDG-CC può essere usato per catturare direttamente e specificamente effettori c-di-GMP dalla miscela complessa di macromolecole come lisati cellulari. Compound Capture basano e approcci proteomica basati chimici sono stati precedentemente segnalati per essere applicabile ad una vasta gamma di organismi, ad esempio Caulobacter crescentus, Salmonella enterica sierotipo typhimurium e P. aeruginosa 1,14. In questo documento metodologico,forniamo una descrizione in profondità della procedura CCMS utilizzando estratti di P. aeruginosa come esempio. Questo studio stabilisce CCMS come uno strumento potente e versatile per identificare biochimicamente nuovi componenti che praticano la piccola molecola di segnalazione.

<table border="0" cellpadding="0" cellspacing="0" width="1179">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">caproBox</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">1-5010-001 (220 V)</td>
			<td style="width:515px;">UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">caproMag</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">included in the CCMS Starter Kit</td>
			<td style="width:515px;">For easy handling of magnetic particles without pipetting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:236px;">c-di-GMP caproKit</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">upon request</td>
			<td style="width:515px;">The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:223px;">GE Healthcare</td>
			<td style="width:207px;">17-0851-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">12-tube PCR strips</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:207px;">AB-1114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UIS250v sonicator with VialTweeter</td>
			<td style="width:223px;">Hielscher ultrasound technology</td>
			<td style="width:207px;">UIS250v and VialTweeter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Miniature French Pressure Cell</td>
			<td style="width:223px;">Thermo Electron Corporation</td>
			<td style="width:207px;">FA-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

capture compound mass spectrometry - a powerful tool to identify novel c-di-gmp effector proteins

Sono stati compiuti notevoli progressi nel corso degli ultimi dieci anni verso l&#39;identificazione e la caratterizzazione di enzimi coinvolti nella sintesi (ciclasi diguanylate) e di degradazione (fosfodiesterasi) del secondo messaggero c-di-GMP. Al contrario, sono disponibili poche informazioni sui meccanismi molecolari e componenti cellulari attraverso cui questa molecola di segnalazione regolamenta una vasta gamma di processi cellulari. La maggior parte delle proteine ​​effettrici noti appartengono alla famiglia Pilz o sono degenerati ciclasi diguanylate o fosfodiesterasi che hanno rinunciato a catalisi e hanno adottato la funzione effettrici. Così, per definire meglio la rete c-di-GMP cellulare in una vasta gamma di batteri metodi sperimentali sono necessari per identificare e convalidare nuovi effettori che affidabile e in silico previsioni FAIL. Abbiamo recentemente sviluppato un nuovo Capture spettrometria di massa Compound (CCMS) tecnologia basata come un potente strumento perbiochimicamente identificare e caratterizzare le proteine ​​di legame c-di-GMP. Questa tecnica è stata precedentemente riportato di essere applicabile ad una vasta gamma di organismi 1. Qui diamo una descrizione dettagliata del protocollo che utilizziamo per sondare tali componenti di segnalazione. Come esempio, si usa Pseudomonas aeruginosa, un patogeno opportunista in cui c-di-GMP gioca un ruolo critico nella virulenza e biofilm controllo. CCMS ha identificato il 74% (38/51) dei componenti note o previste della rete c-di-GMP. Questo studio spiega la procedura CCMS in dettaglio, e stabilisce come uno strumento potente e versatile per identificare nuovi componenti coinvolti in piccola molecola di segnalazione.

Per identificare nuovi c-di-GMP effettori in P. aeruginosa abbiamo usato sistematicamente CCMS per analizzare le frazioni solubili e di membrana di P. aeruginosa ceppo PAO1 da una cultura fase di log (OD 600 = 0,5). Qui riassumiamo e discutere i risultati rappresentativi di questa pesca. Sono stati utilizzati quattro repliche biologiche indipendenti. Per ogni esperimento sono stati utilizzati due differenti concentrazioni CDG-CC (5 micron e 10 micron). Per rilevare se la specificità, esperi...

Watch this Scientific Journal Video about Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins at JoVE.com

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

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 spettrometria di massa Compound - un potente strumento per identificare nuovi c-di-GMP effettori Proteine

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

capture-compound-mass-spectrometry-powerful-tool-to-identify-novel-c

Research

JoVE Journal

Biology

11.5K Views.  Biozentrum of the University of Basel. 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.

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Preparazione del campione: il metodo ultra sottile strato

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

Ricerca

Biologia

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteomica per identificare proteine ​​che interagiscono con P2X2 Ligand-Gated canali di comunicazione

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Una valutazione quantitativa del Lipidome lievito con ionizzazione Electrospray Spettrometria di massa

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitina membrana a base di lievito Due-Hybrid (MITO) Sistema: un potente strumento per identificare interazioni proteina-proteina

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Analizzando complessi proteici mediante spettrometria di massa di grandi dimensioni strutturali

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - un metodo per identificare trascrittoma livello i siti di legame delle proteine ​​RNA binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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

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

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

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

Profilatura di metiltransferasi e altri S-Adenosil- L-Omocisteina-binding proteine ​​mediante spettrometria di massa Compound Capture (CCMS)

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

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Quantificazione delle proteine ​​Utilizzando Arricchimento Peptide immunoaffinità Accoppiato con spettrometria di massa

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Ammide Idrogeno / Deuterio Scambio &amp; massa MALDI-TOF analisi di spettrometria di attivazione Pak2

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

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

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

Profiling tiolo Proteome Redox Utilizzando tag spettrometria di massa isotopica

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