Questo lavoro è supportato in parte dal National Institutes of Health di Grant GM-26738 (a JAT).

1. Pak2 attivazione <ol><li> Aggiungere l&#39;appropriato pre-testati quantità di caspasi 3-20 l di 12 mg / ml Pak2 nel buffer A (50 mM DTT mM Tris-HCl, 150 mM NaCl, e 2 a pH 7,5) e incubare a 34 ° C per 30 min a pieno fendere e attivare Pak2. </li><li> Analizzare i prodotti di scissione mediante SDS-PAGE (10%) e Coommassie colorazione blu. </li><li> Aggiungere il Pak2 spaccati di attivazione del buffer B (50 mM Tris-HCl, 150 mM NaCl, 15 mM MgCl 2, 10 mM ATP) a pH 7,5, in 20 microlitri di una concentrazione finale di 10 mg / ml Pak2, e incubare a 34 ° C per 30 minuti per attivare completamente Pak2 da autofosforilazione a 8 siti. </li><li> Conferma scissione completa di Pak2 dalla migrazione dei frammenti p34 e p27 come determinato dalla SDS-PAGE 10. Un&#39;adeguata verifica delle condizioni autofosforilazione avviene tramite autoradiografia in un esperimento separato con (32 P) ATP o spettrometria di massa per rilevare fosfopeptidi. </li></ol> 2. Identificazionedi pepsina-digerito Pak2 Frammenti <ol><li> Aggiungere 1 ml di 0,1% Acido Trifluroacetic (TFA) a pH 2,5 a lavare le perline agarosio due volte prima dell&#39;uso. Poi, centrifugare i campioni per 15 sec a 2.000 xg per rimuovere le perline. </li><li> Aggiungi Pak2 (20 mg in 2 ml di 150 mM NaCl, 50 mM DTT mM Tris-HCl e 2 a pH 7,5) a 100 ml di 0,1% TFA, e incubare il campione per 5 minuti con 30 ml di pepsina-coniugato perline agarosio . </li><li> Equilibrare una inversione di fase colonna HPLC C18 con un sistema primario di solvente 0,1% TFA a pH 2.5. Il digest pepsina (50 ml) viene eluito con un lineare da 100 min gradiente di 0-80% acetonitrile contenente 0,1% TFA ad un flusso di 0,2 ml / min. Le frazioni vengono raccolti ogni 2 min. </li><li> Asciugare le frazioni HPLC con uno speed-vac (3 h) e risospendere i campioni in una soluzione di acetonitrile (5 mL) e lo 0,1% TFA (5 mL) a pH 2.5. </li><li> Inizialmente, ogni frazione è a screening per peptidi con il MALDI-TOF PerSeptive Biosystems Voyager DE STR (PE Biosystems, Foster City, CA, USA). </li><li> Le 16 frazioni contenenti peptidi sono sottoposti a spettrometria di massa tandem con un Q-TOF spettrometro di massa e un QSTAR XL oMALDI MS / MS per identificare i frammenti. </li><li> La sequenza di ciascun peptide è verificata dagli ioni prodotto generato dalla spettrometria di massa tandem con il teorico m / z coefficienti basati sulla sequenza principale del Pak2 Usando il sito web Protein Prospector ( <a href="http://prospector.ucsf.edu/">http://prospector.ucsf.edu/</a> ) 11. </li></ol> 3. Misura della Amide H / D Cambio <ol><li> Tenere tutte le soluzioni e reagenti necessari per H / D cambio a 25 ° C a bagnomaria. ATP viene disciolto in acqua e il pH è regolato a 7,0 prima dell&#39;uso. L&#39;ulteriore 4mm ATP in D 2 O buffer è di impedire ATP dissociato dal Pak2 dopo la diluizione. </li><li> Avviare H / D scambio con l&#39;aggiunta di 18 ml di tampone D 2 O (50 mM MOPS pH 6,98, 125 mM NaCl) a 2 microlitri (20 mcg) Di Pak2 in un tampone contenente 150 mM NaCl, 50 mM mM Tris-HCl (pH 7,5) e 2 DTT, che comporti un livello Pak2 di 10 mg / ml e un pH di 7,0 a 0 ° C. </li><li> Incubare i campioni in triplice copia in H / D cambio di 0, 0,5, 1, 3, 5 e 10 minuti, o 24 ore </li><li> Estinguere la H / D processo di scambio con l&#39;aggiunta di 180 ml di ghiacciata TFA 0,1% a pH 2.2, che porta il pH a 2,5 in un volume finale di 200 l. Per i campioni contenenti ATP, 180 ml di ghiacciata TFA 0,11% a pH 2.2 è utilizzato per mantenere il pH a 2,5 in stato bonificato. </li><li> Aggiungere un&#39;aliquota (100 l) di ciascun campione spento a 30 ml di pepsina attivata coniugato perline agarosio. La digestione pepsina è autorizzato a procedere su ghiaccio per cinque minuti ed i campioni sono in agitazione ogni 30 sec. </li><li> Terminare la digestione per centrifugazione del campione per 15 sec a 5.000 xga 4 ° C per rimuovere la pepsina. </li><li> Congelare rapidamente i campioni in liquido N 2 e conservare a -80 ° C per non più di 2giorni prima della MALDI-TOF analisi. </li><li> La matrice di MALDI è di 5 mg / ml di α-ciano-4-idrossicinnamici acido (CHCA) disciolto in una soluzione (1:1:1) di acetonitrile, etanolo e 0,1% TFA a pH 2,5 e tenuto a 0 ° C. </li><li> Parzialmente scongelare rapidamente ogni campione e mescolare 1 ml di ogni campione con 1 l di matrice e spot su un 4 ° C Piastra MALDI. </li><li> Applicare un vuoto moderato al piatto ad asciugare i punti in 30 secondi prima di MALDI-TOF analisi. Il tempo tra scongelamento del campione e il recupero dello spettro è di circa 3 min. </li><li> Acquisire MALDI-TOF spettri di massa con un PerSeptive STR Biosystems Voyager DE. Acquisire dati in un 2-GHz, frequenza di campionamento a 100.000 canali di dati, con un 20.000 V tensione di accelerazione, e una tensione di rete del 65% utilizzando l&#39;estrazione con un ritardo di 180 ns ritardo impulsi. Un centinaio di scansioni sono in media di 2 min. Il MALDI-TOF strumento non si limita a qualsiasi marca o software particolari. </li><li> Utilizzare il programma per computer Data Explorer 4.0 per calculmangiato la deuterazione di ogni frammento. </li><li> Calcolare il cambio posteriore in base al rapporto del numero effettivo deuterone incorporata a 24 ore di H / D cambio divisa per tutti i siti possibili intercambiabili, che sono la spina dorsale amidi, tranne N-terminale ammide (Figura 1). </li></ol> 4. Rappresentante dei risultati: La scissione della caspasi e autofosforilazione sono verificati mediante SDS-PAGE colorazione Commassie. Pak2 inattive è una singola banda di 58 kDa. Scissione della caspasi Pak2 è completa, e produce due frammenti, p27 e p34 che migrano separatamente. La migrazione di autophosphorylated p27 e p34 mostra una sovrapposizione sul gel a causa della ritardata migrazione del frammento completamente fosforilati p27 (Figura 2). H / D esperimenti di cambio vengono effettuate più volte tra 0 e 10 min (Figura 3). A titolo di esempio, la durata d&#39;incorporazione deuterio in m / ZPEAK 1697,85 di Pak2 inattivi è composto da più picchi isotopici, come indicato dallo spostamento della busta di massa nel corso del tempo. <img alt="Figura 1" src="/files/ftp_upload/3602/3602fig1.jpg" /> Figura 1. Equazione per il calcolo del deuterazione. <img alt="Figura 2" src="/files/ftp_upload/3602/3602fig2.jpg" /> Figura 2. Autofosforilazione e scissione delle caspasi Pak2. Caspasi-spaccati Pak2 (corsia di sinistra), intatto Pak2 inattiva (corsia centrale) e della caspasi-spaccati autophosphorylated Pak2 (corsia di destra) sono stati analizzati mediante SDS-PAGE e Coommassie colorazione blu. Adattato da Hsu, YH et al 2. <img alt="Figura 3" src="/files/ftp_upload/3602/3602fig3.jpg" /> Figura 3. Spettro di massa di un corso momento della costituzione di deuterio per Pak2 inattivi. Pak2 inattivo è stato sottoposto a H / D SCAMBIOESN per 0-10 min e analizzati da MALDI-TOF. Un esempio della distribuzione isotopica del ampliato MALDI-TOF di picco m / z 1697,85 durante il periodo di tempo di H / D scambio è mostrato. La linea rossa tratteggiata indica la media della busta di massa e lo spostamento della busta di massa mostra il deuterazione di un peptide.

<ol>
	<li>Anand, G. S., Hughes, C. A., Jones, J. M., Taylor, S. S., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+H/2H+exchange+reveals+communication+between+the+cAMP+and+catalytic+subunit-binding+sites+in+the+R(I)alpha+subunit+of+protein+kinase.">Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase.</a> A. J. Mol. Biol. 323, 377-386 (2002).</li><li>Hsu, Y. H., Johnson, D. A., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+conformational+changes+during+activation+of+protein+kinase+Pak2+by+amide+hydrogen/deuterium+exchange.">Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange.</a> J. Biol. Chem. 283, 36397-36405 (2008).</li><li>Englander, S. W., Kallenbach, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+and+structural+dynamics+of+proteins+and+nucleic+acids.">Hydrogen exchange and structural dynamics of proteins and nucleic acids.</a> Q. Rev. Biophys. 16, 521-655 (1983).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Commun. Mass. Spectrom. 5, 214-217 (1991).</li><li>Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+the+membrane+binding+region+of+Group+VIA+Ca2+-independent+phospholipase+A2+using+peptide+amide+hydrogen/deuterium+exchange+mass+spectrometry.">Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 284, 23652-23661 (2009).</li><li>Hsu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+rigidifies+the+C2+domain+and+the+intradomain+interaction+of+GIVA+phospholipase+A2+as+revealed+by+hydrogen/deuterium+exchange+mass+spectrometry.">Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 283, 9820-9827 (2008).</li><li>Lee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hPAK65+by+caspase+cleavage+induces+some+of+the+morphological+and+biochemical+changes+of+apoptosis.">Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis.</a> Proc. Natl. Acad. Sci. U. S. A. 94, 13642-13647 (1997).</li><li>Rudel, T., Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+morphological+changes+in+apoptotic+cells+regulated+by+caspase-mediated+activation+of+PAK2.">Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2.</a> Science. 276, 1571-1574 (1997).</li><li>Roig, J., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytostatic+p21+G+protein-activated+protein+kinase+gamma-PAK.">Cytostatic p21 G protein-activated protein kinase gamma-PAK.</a> Vitam. Horm. 62, 167-198 (2001).</li><li>Walter, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+and+activation+of+p21-activated+protein+kinase+gamma-PAK+by+CPP32+(caspase+3).+Effects+of+autophosphorylation+on+activity.">Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity.</a> J. Biol. Chem. 273, 28733-28739 (1998).</li><li>Clauser, K. R., Baker, P., Burlingame, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+accurate+mass+measurement+(+/-+10+ppm)+in+protein+identification+strategies+employing+MS+or+MS/MS+and+database+searching.">Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.</a> Anal. Chem. 71, 2871-2882 (1999).</li></ol>

Nessun conflitto di interessi dichiarati.

Identificazione della pepsina-digerito Frammenti è un passo fondamentale della H / D esperimento di scambio. Identificazione errata del peptide può condurre a conclusioni errate. Pak2 pepsina-digerito viene eluito in una colonna HPLC in fase inversa C18 per ottenere un fondo pulito. Nei nostri esperimenti, un totale di 40 frazioni sono state raccolte e sottoposte a MALDI-TOF. Peptidi multipli potrebbero essere identificati in ciascuna delle frazioni. I peptidi sono stati sottoposti a spettrometria di massa tandem (Q-TOF MS / MS e oMALDI MS / MS) per identificare le loro sequenze. La MS / MS spettri di un peptide dovrebbe avere almeno tre ioni prodotto per confermare l&#39;identificazione del peptide. Il programma per computer Data Explorer 4.0 (Applied Biosystems) esegue la correzione iniziale di base e filtraggio del rumore. Ogni spettro deve essere calibrata sulla base di due picchi in sequenza. Noi calibrare il nostro spettro da parte della massa teorica del undeuterated m / z che sono 923,45 e 1697,84. Tlui accuratezza di massa dopo la calibrazione può raggiungere i 10 ppm. Su deuterazione, il mono-isotopo può passare ad una massa maggiore. Aumenta passo unitario a questi m / z rapporti ceduto le masse associate a una maggiore deuterazione. L&#39;intensità dei picchi della busta di massa non influisce sul livello deuterazione. Tuttavia, l&#39;intensità dei picchi dei picchi isotopici in una busta di massa particolare sono fondamentali per il calcolo della massa media. Il primo passo del calcolo del deutone è incorporato sottraendo la massa del peptide non deuterati campione dal campione deuterato. Tre altri fattori, la diluizione D 2 O, il deuteroni residuo nelle catene laterali e il cambio posteriore, dovranno essere ulteriormente considerati nel calcolo (Figura 1). La diluizione D 2 O è il fattore di diluizione D 2 O dopo la miscelazione del campione e D 2 O di buffer di avviare la H / D cambio. Deutone residua (4,5%) nelle catene laterali della pepsina-digerirepeptidi ed ha bisogno di essere sottratto. Il back-scambio è l&#39;inevitabile perdita di deuterazione in tutti i peptidi deuterato e pepsina-digerito nel processo di misura di massa. Il peptide più accessibile solvente (m / z = 1105,60) dopo 24 ore di H / D cambio rappresenta una regione ricca deuterazione. Il numero deuterone incorporato per ciascuno dei peptidi è la differenza tra il baricentro dei peptidi deuterati e non deuterati peptica Pak2. Il più grande deuterati peptide m / z 1105 è stato selezionato per rappresentare deuterazione piena quando Pak2 era a 24 ore di H / D cambio. Il Rapporto di Cambio posteriore è stato calcolato il numero effettivo deuterone incorporata a 24 ore di H / D cambio divisa per tutti i siti possibili scambiabile a peptide m / z 1105.

<table border="1"></table><table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> D 2 O </td><td> Aldrich </td><td> 7789-20-0 </td><td></td></tr><tr><td> α-ciano-4-acido idrossicinnamici </td><td> Sigma </td><td> 28166-41-8 </td><td></td></tr><tr><td> MALDI-TOF </td><td> PE Biosystems </td><td> Voyager DE STR </td><td></td></tr><tr><td> Q-TOF Spettrometro di massa </td><td> Q-TOF Ultima-Global </td><td></td><td></td></tr><tr><td> QSTAR XL oMALDI MS / MS </td><td> Applied Biosystems </td><td></td><td></td></tr><tr><td> Dati Explorer 4.0 programma per computer </td><td> Applied Biosystems </td><td></td><td></td></tr&gt; </tbody></table>

amide hydrogen/deuterium exchange &amp; maldi-tof mass spectrometry analysis of pak2 activation

Ammide idrogeno / deuterio scambio (H / D cambio) accoppiata a spettrometria di massa è stata ampiamente utilizzata per analizzare l&#39;interfaccia di interazioni proteina-proteina, i cambiamenti conformazionali delle proteine, la dinamica delle proteine ​​e le interazioni proteina-ligando. H / D cambio sulle posizioni ammidico la spina dorsale è stata utilizzata per misurare i tassi deuterazione delle micro-regioni di una proteina mediante spettrometria di massa 1,2,3. La risoluzione di questo metodo dipende dalla digestione pepsina deuterati della proteina di interesse in peptidi che normalmente gamma 3-20 residui. Anche se la risoluzione di H / D cambio misurato mediante spettrometria di massa è inferiore alla risoluzione singolo residuo misurata dal eteronucleari singola coerenza quantistica (HSQC) metodo di NMR, la misurazione della spettrometria di massa in H / D cambio non è limitata dalla dimensione del proteine ​​4. H / D lo scambio avviene in soluzione acquosa che mantiene la conformazione delle proteine. Mettiamo a disposizione un metodo che utilIZES il MALDI-TOF per la rilevazione di 2, al posto di un HPLC / ESI (ionizzazione electrospray)-MS sistema di 5,6. Il MALDI-TOF fornisce dati accurati intensità di massa per i peptidi della proteina digerita, in questo caso Pak2 proteina chinasi (detta anche γ-Pak). Proteolisi di Pak 2 è effettuato in una digestione pepsina non in linea. Questo metodo alternativo, quando l&#39;utente non ha accesso a una colonna HPLC e pepsina collegato alla spettrometria di massa, o quando la colonna pepsina in HPLC non si traduca in una mappa digestione ottimale, per esempio, il pesante disolfuro-bonded secreto fosfolipasi A 2 (SPLA 2). Utilizzando questo metodo, abbiamo monitorato con successo i cambiamenti nel livello deuterazione durante l&#39;attivazione della caspasi 3 Pak2 di scissione e autofosforilazione 7,8,9.

Watch this Scientific Journal Video about Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation at JoVE.com

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

MALDI-TOF spettrometria di massa è stato utilizzato con successo per monitorare la amide idrogeno / deuterio in cambio di attivazione della proteina chinasi Pak2.

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 ...

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Biology

17.7K Views.  Tunghai University. MALDI-TOF spettrometria di massa è stato utilizzato con successo per monitorare la amide idrogeno / deuterio in cambio di attivazione della proteina chinasi Pak2.

Video: Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

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

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 ...

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. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analisi diretta di singole cellule mediante spettrometria di massa a pressione atmosferica

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

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 ...

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

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

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

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

L&#39;uso di MALDI-TOF spettrometria di massa e un database personalizzato per caratterizzare batteri indigeni ad un unico ambiente di Cave (Kartchner Caverns, AZ, USA)

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

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

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

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

Reazione selezionato Monitoraggio spettrometria di massa per Absolute Protein Quantificazione

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

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

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

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

Arricchimento della matrice extracellulare proteine ​​da tessuti e digestione in Peptidi per l&#39;analisi Spettrometria di Massa

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

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Applicazioni del singolo-probe: Mass Spectrometry Imaging e analisi singola cella in condizioni ambientali

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Flusso di lavoro completo per l&#39;analisi dell&#39;istone modificazioni post-traduzionali Utilizzando Bottom-up Spettrometria di Massa: Da istone estrazione all&#39;analisi dei dati

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...