Gli autori sono grati per il sostegno finanziario di questo lavoro dalla US National Science Foundation sotto Grant No. 0719232, dal Dipartimento dell&#39;Energia degli Stati Uniti (DEFG02-01ER15129), e da Protea Biosciences, Inc. (Morgantown, WV). Gli autori desiderano inoltre ringraziare Jessica A. Stolee per il suo aiuto durante la videoregistrazione del protocollo.

Il protocollo che segue descrive i passi principali della ionizzazione elettrospray ablazione laser (LAESI) esperimento e fornisce esempi rappresentativi per la laterale e tridimensionali (3D), immagini di animali e campioni di tessuti vegetali. Ulteriori dettagli sperimentali e tecniche può essere ottenuto da altre parti. 1-6 1. Preparazione e montaggio del tessuto <ol><li> Se sezionamento è necessario, utilizzare un cryomicrotome alla sezione del tessuto in 10-100 micron fette spesse a -10 a -20 ° C se non diversamente indicato per un tipo di tessuto particolare. </li><li> Monte sezioni su una superficie piana (ad esempio, chimicamente pre-pulito vetrino) direttamente, senza modificatori chimici. Per i tessuti sezionati, disgelo con montaggio delle sezioni e sicuro supporto del campione ad un Peltier di raffreddamento stadio subito dopo disgelo montaggio per mantenere il tessuto congelato in ogni momento durante l&#39;analisi. Questo passaggio è necessario per ridurre al minimo / prevenire la migrazione molecolare della sezione. </li><li> Se necessario, utilizzare un dissipatore dotato di una ventola a basso consumo per facilitare la rimozione del calore dal Peltier stadio per mantenere il tessuto congelato. </li><li> In un ambiente umido per un lungo periodo di tempo (1-2 ore), per controllare condensazione di acqua o ghiaccio sulla superficie del tessuto. Condensa sul tessuto influenza negativamente le prestazioni di imaging in esperimenti LAESI 1. </li><li> Se necessario, utilizzare un deumidificatore stanza o posto il campione raffreddato in una camera climatica riempito con un gas inerte (ad esempio, gas azoto secco) per evitare la condensa 1. </li></ol> 2. Ottimizzazione della sorgente di ioni LAESI La sorgente di ioni LAESI consiste in un mid-laser infrarosso, una serie di elementi ottici per la direzione della luce e messa a fuoco così come i titolari campione supplementare, componenti di raffreddamento, le fasi di traduzione, e un sistema di erogazione del solvente. La Figura 1 mostra la disposizione tipica di questi elementi rispetto ai all&#39;ingresso della pressione atmosferica sorgente ionica di uno spettrometro di massa. <ol><li> Come illustrato nella figura 1, la posizione del campione di 15-20 millimetri sotto l&#39;orifizio del cono di campionamento spettrometro di massa (d O-FP). </li><li> Operare un mid-IR laser a lunghezza d&#39;onda 2,94 micron e 10 Hz frequenza di ripetizione. Attenuare la potenza laser a ~ 100 μJ / impulsi di energia. </li><li> Utilizzare una combinazione di specchi d&#39;oro e una lente di messa a fuoco trasparente alla lunghezza d&#39;onda laser (per esempio, un piano-convessa CaF 2 o ZnSe lente) per accoppiare la energia dell&#39;impulso laser nel campione di incidenza normale (vedi incidenza con angolo retto rispetto alla superficie del campione nella Figura 1). </li><li> Posizionare il medio infrarosso asse fascio 5-8 mm di fronte l&#39;orifizio del cono di campionamento dello spettrometro di massa. </li><li> Regolare la posizione della lente di messa a fuoco e l&#39;energia degli impulsi del raggio laser per ottenere la rimozione dei tessuti nel punto focale. Le dimensioni del volume ablato determinare il pixel (o voxel in immagini tridimensionali) dimensioni per l&#39;applicazione di imaging. </li><li> Posizionare un emettitore nanospray in linea con l&#39;asse di ingresso dello spettrometro di massa e in un orifizio a emettitore distanza punta di ~ 10 mm (vedi figura 1). </li><li> Per la electrospray, preparare una soluzione metanolo 50% con 0,1% di acido acetico o 0,1% di additivo acetato di ammonio per la modalità di ioni positivi o negativi, rispettivamente. A seconda del campione, altri solventi organici, quali acetonitrile, isopropanolo, ecc, può sostituire il metanolo a concentrazioni appropriate per l&#39;attività di analisi. La stabilità del electrospray è cruciale per l&#39;imaging di successo. In base alla selezione solvente, la portata e la tensione spruzzo devono essere adeguate per raggiungere spruzzo stabile. </li><li> Per reattivi LAESI 6 in applicazioni di imaging, la soluzione electrosprayed può contenere reagenti. </li><li> Utilizzare una pompa a siringa per fornire la soluzione electrospray attraverso l&#39;emettitore electrospray ad una portata di circa 300 Nl / min. </li><li> Se l&#39;orifizio spettrometro di massa viene mantenuta a bassa tensione (&lt;500 V misurato contro terra), generare electrospray applicando alta tensione direttamente al emettitore elettrospray (ad esempio, 3000 V) o attraverso un raccordo metallico. Altrimenti, terra direttamente l&#39;emettitore electrospray o mediante l&#39;unione di metallo per stabilire elettrospray. </li><li> Attivare la sorgente electrospray a cono-nebulizzazione modalità per la generazione di ioni più efficiente per LAESI. Per l&#39;effetto di variabili operative sulle modalità di spruzzatura e il loro effetto sulla spettri di massa, vedi discussioni altrove. 5,7,8 </li><li> Regolare con cura le distanze relative del setup LAESI per ottimizzare rendimento LAESI ioni mantenendo il fascio laser, l&#39;emettitore, e gli assi orifizio sullo stesso piano. Si prega di trovare le istruzioni dettagliate altrove 5. </li><li> Con un microscopio ottico, determinare le dimensioni laterali del cratere ablazione sul campione. </li><li> Per tridimensionale esperimenti di imaging LAESI, eseguire l&#39;ablazione con impulsi individuali e di determinare la profondità di un voxel utilizzando, per esempio, lo z-stack mode in microscopia ottica 3. </li></ol> 3. Imaging molecolare e analisi dei dati Nell&#39;esperimento di imaging, il campione di tessuto viene spostato nel piano focale del laser nelle direzioni X e Y con dimensioni passo maggiore o uguale alle dimensioni dello spot ablazione. La risoluzione spaziale è limitata dalla focalizzazione del fascio laser incidente. <ol><li> Selezionare l&#39;area di interesse sulla superficie del campione e ottenere la (X, Y) coordinate dei confini corrispondente. </li><li> Ha scelto un algoritmo gridding (ad esempio, griglia adattiva, l&#39;imaging regione selezionata, griglia rettangolare, modello a spirale, Z scansione, ecc) con cui raster superficie del campione selezionato con tempo di permanenza per ogni pixel sopra l&#39;area da acquisire. </li><li> Utilizzare un tre assi fase di traduzione e software che è in grado di rastering il campione secondo la griglia predeterminata. </li><li> Calcolare il tempo totale richiesto per l&#39;imaging. </li><li> Disabilitare il limite di acquisizione dati in tempo dello spettrometro di massa. Se questo non è possibile, impostare il limite di acquisizione dati in tempo al valore calcolato il tempo di imaging. </li><li> Avviare il medio infrarosso sorgente laser ad una frequenza di ripetizione corretta di produrre sufficiente rapporto segnale-rumore nello spettro di massa entro il tempo di permanenza per ogni pixel per eseguire un esperimento di imaging LAESI laterale. Per il 3D imaging molecolare, utilizzano una frequenza di acquisizione dello spettro superiore al tasso di ripetizione sorgente laser al successo di massa analizzare gli ioni generati all&#39;interno di un singolo impulso laser. Attendere il segnale di START per avviare la sequenza di ablazione. </li><li> Accendere la sorgente electrospray. Assicurarsi che ci sia una soluzione sufficiente per il tempo pieno richiesto per l&#39;imaging. </li><li> Contemporaneamente avviare l&#39;acquisizione di spettri di massa, alla metà degli IR ablazione laser, e la scansione della superficie. </li><li> Quando la corsa di imaging ha finito, STOP la scansione della superficie, la metà degli IR laser, e l&#39;acquisizione dei dati. </li><li> Disabilitare la sorgente laser. </li><li> Spegnere l&#39;alta tensione. </li><li> Spegnere la pompa a siringa. </li><li> Impostare lo spettrometro di massa in modalità Standby. </li><li> Spegnere il Peltier di raffreddamento dell&#39;elettronica. </li><li> Chiudere il flusso di gas inerte se usato. </li><li> Utilizzare un software di correlare le coordinate assolute dei pixel in immagini laterale o il voxel in analisi 3D con gli spettri corrispondenti. </li><li> Tracciare il segnale di intensità di ioni per un selezionato m / z valore contro le coordinate assolute di analisi per ottenere immagini molecolari laterale e 3D. </li></ol> 4. Rappresentante Risultati La Figura 2 dà risultati rappresentativi per alcuni tipi di tessuti importanti e modalità di imaging. Un pannello descrive un caso di una sezione di tessuto animale che è stato congelato durante l&#39;esperimento per prevenire la disidratazione. 1 In aggiunta, il campione si trovava in un ambiente asciutto gas azoto per evitare i vapori d&#39;acqua ambiente da condensa sulla superficie del campione. Un 100-micron di spessore sezione coronale di un cervello di ratto (Rattus norvegicus) è stato ripreso con LAESI lateralmente. Le regioni anatomiche del cervello (vedi immagine ottico in pannello A) mostrano una buona correlazione con l&#39;immagine molecolare ottenuto per il PC plasmalogeni (O-33: 3) e / o PE (O-36: 3) con m / z 728,559. Pannello B mostra l&#39;imaging 3D LAESI di un impianto di Zebra (Aphelandra squarrosa) del tessuto fogliare. Perché lascia possedere un meccanismo naturale di difesa contro la disidratazione, il campione può essere interrogato per l&#39;ambiente circostante. 3 Il ottenute immagini 3D molecolare ha rivelato una serie di modelli di distribuzione per i metaboliti primari e secondari. Tra gli altri, acacetin con m / z 285,076 è stato rilevato al conteggio di ioni superiore nei settori giallo degli strati secondo e il terzo dalla parte superiore con una distribuzione omogenea nelle altre. Questa distribuzione d&#39;accordo con il modello della variegatura visto in ottica dell&#39;immagine. <img src="/files/ftp_upload/2097/2097fig1.jpg" alt="Figura 1" /> Figura 1. Schematica del sistema LAESI (ES, punta emettitore electrospray; O, orifizio del cono di campionamento dello spettrometro di massa, FL, concentrandosi lente, FP, punto focale, P, Peltier fase di raffreddamento; HS, dissipatore di calore). Una parte del materiale particolato espulso durante la metà degli IR ablazione (punti rossi) si fonde con la elettrospray per produrre gocce carica seminato con molecole e ioni del campione (punti verdi). Gli ioni liberati da queste gocce sono analizzati e registrati dal spettrometro di massa. <img src="/files/ftp_upload/2097/2097fig2.jpg" alt="Figura 2" /> Figura 2. Risultati rappresentativi per la laterale e 3D immaginario. LAESI ng con spettrometria di massa (A) Il pannello superiore mostra l&#39;immagine ottica di un cervello di ratto (Rattus norvegicus) sezione coronale e l&#39;immagine molecolare ottenuto per il PC plasmalogeni (O-33: 3) e / o PE (O-36 : 3) con m / z 728,559. La barra di scala bianca corrisponde a 1 mm. Adattato con il permesso (Riferimento 1). Copyright 2010 American Chemical Society. (B) Il pannello in basso mostra l&#39;imaging 3D di una foglia da un variegato Zebra pianta (Aphelandra squarrosa). Acacetin con m / z 285,076 è stato rilevato al conteggio di ioni superiore nei settori giallo degli strati secondo e il terzo dalla parte superiore con una distribuzione omogenea nelle altre. Ristampato con il permesso (Riferimento 3). Copyright 2009 American Chemical Society.

<ol>
	<li>Nemes, P., Woods, A. S., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Imaging+of+Small+Metabolites+and+Lipids+in+Rat+Brain+Tissues+at+Atmospheric-Pressure+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Simultaneous Imaging of Small Metabolites and Lipids in Rat Brain Tissues at Atmospheric-Pressure by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Anal Chem. 82, 982-988 (2010).</li><li>Nemes, P., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+electrospray+ionization+for+atmospheric-pressure,+in+vivo,+and+imaging+mass+spectrometry.">Laser ablation electrospray ionization for atmospheric-pressure, in vivo, and imaging mass spectrometry.</a> Anal Chem. 79, 8098-8106 (2007).</li><li>Nemes, P., Barton, A. A., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+metabolites+in+tissues+under+ambient+conditions+by+laser+ablation+electrospray+ionization+mass+spectrometry.">Three-dimensional imaging of metabolites in tissues under ambient conditions by laser ablation electrospray ionization mass spectrometry.</a> Anal Chem. 81, 6668-6675 (2009).</li><li>Nemes, P., Barton, A. A., Li, Y., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+molecular+imaging+and+depth+profiling+of+live+tissue+by+infrared+laser+ablation+electrospray+ionization+mass+spectrometry.">Ambient molecular imaging and depth profiling of live tissue by infrared laser ablation electrospray ionization mass spectrometry.</a> Anal Chem. 80, 4575-4582 (2008).</li><li>Nemes, P., Vertes, A., Nemes, S. S., Rubakhin, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+electrospray+ionization+for+atmospheric-pressure+molecular+imaging+mass+spectrometry.+Mass+Spectrometry+Imaging.">Laser ablation electrospray ionization for atmospheric-pressure molecular imaging mass spectrometry. Mass Spectrometry Imaging.</a> Methods in Molecular Biology. , (2010).</li><li>Shrestha, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+lipids+and+small+metabolites+in+mouse+brain+tissue+by+AP+IR-MALDI+and+reactive+LAESI+mass+spectrometry.">Direct analysis of lipids and small metabolites in mouse brain tissue by AP IR-MALDI and reactive LAESI mass spectrometry.</a> Analyst. 135, 751-758 (2010).</li><li>Nemes, P., Marginean, I., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spraying+mode+effect+on+droplet+formation+and+ion+chemistry+in+electrosprays.">Spraying mode effect on droplet formation and ion chemistry in electrosprays.</a> Anal Chem. 79, 3105-3116 (2007).</li><li>Nemes, P., Goyal, S., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+and+noncovalent+complexation+changes+in+proteins+during+electrospray+ionization.">Conformational and noncovalent complexation changes in proteins during electrospray ionization.</a> Anal Chem. 80, 387-395 (2008).</li><li>Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+by+Mid-IR+laser+ablation+mass+spectrometry.">Molecular imaging by Mid-IR laser ablation mass spectrometry.</a> Appl Phys A-Mater Sci Process. 93, 885-891 (2008).</li><li>Shrestha, B., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+metabolic+profiling+of+single+cells+by+laser+ablation+electrospray+ionization+mass+spectrometry.">In situ metabolic profiling of single cells by laser ablation electrospray ionization mass spectrometry.</a> Anal Chem. 81, 8265-8271 (2009).</li><li>Cooks, R. G., Ouyang, Z., Takats, Z., Wiseman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+mass+spectrometry.">Ambient mass spectrometry.</a> Science. 311, 1566-1570 (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

Tipi di tessuto differenti mostrano diversi contenuto d&#39;acqua e resistenza alla trazione, che, a loro volta, possono influenzare le caratteristiche di ablazione dei campioni. 9 Per mitigare questi effetti, si desidera che i protocolli la fluenza laser, manipolazione del campione, analisi e revisione quando si passa da principali tipi di tessuto. Per singola cella o indagini ad alta risoluzione, il medio infrarosso della luce può essere accoppiato in una fibra ottica affilato invece di una lente di focalizzazione. 10 Con il posizionamento della punta della fibra nelle immediate vicinanze delle celle selezionate in un tessuto, LAESI analisi può essere eseguita su uno livello di singola cellula. Come un marchio senza sorgente di ioni per la spettrometria di massa a ionizzazione ambiente, 11 LAESI ha dimostrato un grande potenziale per lo studio dei processi biochimici nei tessuti. Con i benefici aggiunti di analisi diretta, l&#39;imaging laterale e 3D, LAESI è uno strumento emergente bioanalitici per il profiling così come le applicazioni di imaging.

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<td>Mass spectrometer</td>
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<td>Mid-IR laser</td>
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atmospheric-pressure molecular imaging of biological tissues and biofilms by laesi mass spectrometry

Metodi di ionizzazione ambiente in spettrometria di massa permettono indagini analitiche da effettuare direttamente su un tessuto o biofilm in condizioni sperimentali di tipo nativo. Ionizzazione ablazione laser elettrospray (LAESI) ne è un esempio ed è particolarmente adatto per lo studio di acque contenenti i campioni. LAESI utilizza un medio infrarosso raggio laser (lunghezza d&#39;onda 2,94 micron) per eccitare le molecole d&#39;acqua del campione. Quando la soglia fluenza ablazione viene superato, il materiale del campione viene espulsa sotto forma di particolato e di questi proiettili viaggio a decine di millimetri al di sopra della superficie del campione. In LAESI, questo pennacchio ablazione viene intercettato da goccioline molto carico per catturare una frazione del materiale del campione espulso e convertire i suoi costituenti chimici in fase gassosa ioni. Uno spettrometro di massa equipaggiato con un pressione atmosferica interfaccia sorgente ionica è utilizzata per analizzare e registrare la composizione degli ioni liberati provenienti dalla zona sondato (pixel) del campione. Un interrogatorio sistematico su un array di pixel apre una via per l&#39;imaging molecolare in modalità di analisi microsonda. Un aspetto unico di imaging LAESI spettrometria di massa è profiling profondità che, in combinazione con immagini laterali, consente tridimensionale (3D) l&#39;imaging molecolare. Con gli attuali risoluzioni laterali e la profondità di circa 100 micron e ~ 40 micron, rispettivamente, l&#39;imaging LAESI spettrometria di massa consente di esplorare la struttura molecolare dei tessuti biologici. Qui, passiamo in rassegna i principali elementi di un sistema LAESI e fornire linee guida per un esperimento di imaging di successo.

Watch this Scientific Journal Video about Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry at JoVE.com

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ionizzazione ablazione laser elettrospray (LAESI) è una pressione atmosferica sorgente di ioni per la spettrometria di massa. Nella modalità di imaging, un medio infrarosso sonde laser le distribuzioni delle molecole attraverso una sezione di tessuto o di un biofilm. Questa tecnica presenta un nuovo approccio per diversi studi bioanalitici effettuata sotto natale condizioni sperimentali.

Pressione atmosferica Imaging molecolare di tessuti biologici e biofilm da LAESI Spettrometria di Massa

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

atmospheric-pressure-molecular-imaging-biological-tissues-biofilms

Research

JoVE Journal

Biology

14.1K Views.  George Washington University. Ionizzazione ablazione laser elettrospray (LAESI) è una pressione atmosferica sorgente di ioni per la spettrometria di massa. Nella modalità di imaging, un medio infrarosso sonde laser le distribuzioni delle molecole attraverso una sezione di tessuto o di un biofilm. Questa tecnica presenta un nuovo approccio per diversi studi bioanalitici effettuata sotto natale condizioni sperimentali.

Video: Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

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 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 ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

Quantitativa localizzazione di una proteina di Golgi da Imaging sua massa centro di fluorescenza

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...