Gli autori riconoscono sostegno finanziario dalle seguenti istituzioni: la National Science Foundation (Grant 0.719.232), il Dipartimento dell&#39;Energia (Grant DEFG02-01ER15129), il WM Keck Foundation (Grant 041.904), e la George Washington University Fondo Valorizzazione della Ricerca. Gli autori sono grati per le fibre ottiche GeO2 basato generosamente forniti da Fiber Systems infrarossi (Silver Spring, MD), così come per la discussione iniziale sul protocollo relativo alle fibre di incisione da Mark E. Reeves e Joan A. Hoffmann della George Washington Università. Gli autori desiderano inoltre ringraziare Jessica A. Stolee per il suo aiuto durante la videoregistrazione del protocollo.

1. I componenti ottici <ol><li> Ablazione laser per la ionizzazione elettrospray ablazione laser (LAESI) spettrometria di massa (MS) è prodotto da un ns 5 impulso laser a lunghezza d&#39;onda 2,94 micron. Un sintonizzabile oscillatore parametrico ottico guidato da un laser Nd: YAG (Opolette 100, Opotek Inc., Carlsbad, CA) è utilizzata per l&#39;ablazione in questo studio. Il tasso di ripetizione è stato selezionato tra i 5 ei 100 Hz.. Si tratta di un laser di classe IV, che in caso di esposizione diretta può causare gravi danni permanenti agli occhi e alla pelle. La riflessione diffusa del raggio laser può anche essere pericolosa per la pelle o degli occhi. Indossare un adeguato laser occhiali di protezione. </li><li> La fibra ottica di vetro a base di biossido di germanio è stato ottenuto da infrarossi Fiber Systems, Inc. (Silver Spring, MD). Se ci sono Hytrel e poliimmide rivestimenti in fibra di seguire le indicazioni qui sotto per rimuoverli dalle estremità. Calore 1-metil-2-pirrolidone a 130-150 ° C in una cappa aspirante. Immergere le estremità della fibra si vuole mettere a nudo in questo liquido per circa 1 minuto o fino a quando il rivestimento comincia ad ammorbidirsi e staccare. Immergere il rivestimento ammorbidito in metanolo o isopropanolo per un minuto, ed eliminare ogni residuo di rivestimento con un panno privo di tovagliolo. Dopo aver rimosso il rivestimento, fendere entrambe le estremità della fibra con una lama di zaffiro (Kitco Fibra Ottica, Virginia Beach, VA), segnando e delicatamente scattare. </li><li> Per ottenere la nitidezza desiderata, chimicamente etch una estremità della punta della fibra di 1% (v / v) di acido nitrico (grado reagente) soluzione. Verticalmente immergere l&#39;estremità della fibra selezionati nella soluzione acida ad una profondità tra i 300 ei 500 micron, dopo il contatto iniziale. Dopo circa 15 minuti di incisione, la punta si staccano spontaneamente dalla superficie acido. Ciò si traduce in una punta affilata con fibra ~ 15 micron raggio di curvatura. Risciacquare con acqua deionizzata per rimuovere eventuali residui di acido. </li><li> Montare la fine della fibra inciso su un micromanipolatore (MN-151, Narishige, Tokyo, Giappone) e portarlo alla vicinanza (~ 20 micron) della superficie cellulare per la deposizione di efficienza energetica. </li><li> Tenere la non incise fine della fibra ottica da una fibra mandrino nudo (BFC300, Siskiyou Corporation, Grants Pass, OR). Per l&#39;allineamento ottico, la fibra mandrino è montato su un cinque assi traduttore (BFT-5, Siskiyou Corporation, Grants Pass, OR). L&#39;energia laser (da 0,5 a 1 mJ) è accoppiato in fibra mettendo a fuoco il fascio di un piano-convessa fluoruro di calcio o di lenti antiriflesso ZnSe patinata (Infrared prodotti ottici, Farmingdale, NY) sulla sua non-inciso fine. Specchi d&#39;oro protette (PF10-03-M01, Thorlabs, Newton, NJ) sono utilizzati per indirizzare la metà degli IR raggio laser sulla lente di messa a fuoco. </li></ol> 2. Elettrospray installazione <ol><li> Il sistema elettrospray può essere costruito utilizzando i seguenti componenti: l&#39;unione in metallo con ghiera perfluoroelastomero conduttivo, raccordi, tubi guaina, porta aghi (ad esempio, U-435, M215, F-331Nx, F-242x, 9013, rispettivamente IDEX Salute e delle Scienze, Oak Harbor, WA), così come tubi di silice fusa (ad esempio, CT360-100-50-5, Nuovo Obiettivo Inc., Woburn, MA). L&#39;emettitore raccomandata è realizzato in acciaio inossidabile ed ha una punta conica con una id 50 micron (ad esempio, MT320-50-5-5, Nuovo Obiettivo Inc., Woburn, MA). </li><li> Preparare la soluzione di metanolo 50% con 0,1% di acido acetico (v / v) per la elettrospray. La soluzione viene pompata ad una velocità da 200 a 300 Nl / min con una pompa a siringa (Harvard Apparatus, Holliston, MA) utilizzando, ad esempio, una siringa da 500 microlitri (81222, Società di Hamilton, Reno, NV). </li><li> Applicare ad alta tensione tra 2.800 e 3.000 V per il sindacato di metallo da un alimentatore stabilizzato (PS350, Stanford Research Systems Inc, Sunnyvale, CA) per generare un elettrospray costante. Assicurarsi che tutte le connessioni elettriche sono sicure e schermati con la schermatura messa a terra. Il contatto diretto con esposti ad alta tensione può causare scosse elettriche. </li></ol> 3. Esempio di Manipolazione <ol><li> Ottenere il campione di tessuto vivo o cellule da una fonte appropriata e tenerli come raccomandato prima dell&#39;analisi. Le lampadine Allium cepa per questa manifestazione sono stati acquistati da un negozio locale. Tagliare una lampadina longitudinalmente da una bisturi chirurgico. Un segmento di pochi centimetri di una scala è stato sezionato in una striscia. Un monostrato intatto il tessuto epidermico interno è staccata. </li><li> La superficie bagnata dell&#39;epidermide è usato per montare il tessuto su un vetrino pre-lavaggio microscopio. Tenere la diapositiva da una piastra di supporto generale scopo (FP01, Thorlabs Inc., Newton, NJ) montata su un tre assi fase di traduzione per il posizionamento. Acquisire gli spettri di massa subito dopo il montaggio del campione per evitare la degradazione delle cellule. </li></ol> 4. Sistema di visualizzazione <ol><li> Due microscopi a lunga distanza sono usati per selezionare le celle di analisi e di mantenere la distanza tra la punta della fibra affilata e la superficie cellulare. Questo è montato sotto i 15 ° -30 ° angolo misurato dalla superficie. Questo sistema consiste di almicroscopio distanza ong video (zoom ottico 7x precisione, Edmund Optics, Barrington, New Jersey), con un infinito 5x corretto lente dell&#39;obiettivo (Apo Piano M 5x, Mitutoyo Co., Kanagawa, Giappone) dotato di una fotocamera CCD (Marlin F131, Allied Vision Technologies , Stadtroda, Germania) per il monitoraggio e la cattura delle immagini. </li><li> Il microscopio secondo video è montato ad angolo retto rispetto alla superficie del campione per visualizzare le cellule dall&#39;alto. Questo sistema fornisce un feedback visivo per allineare la punta della fibra sopra la cella selezionata per l&#39;ablazione e l&#39;analisi. Si tratta di una precisione dello zoom ottico 7x (Edmund Optics, Barrington, New Jersey), dotato di un infinito 10x corretto lungo lavoro lente dell&#39;obiettivo distanza (M Piano 10x Apo, Mitutoyo Co., Kanagawa, Giappone) e una camera CCD. </li></ol> 5. Acquisizione di spettri di massa <ol><li> Accendere il laser, il electrospray e lo spettrometro di massa, ad esempio, un quadrupolo tempo di volo del sistema (Q-TOF Premier, Waters, Milford, MA). Ottimizzare la geometria della sorgente di ioni LAESI regolando la posizione della macchia ablazione ed emettitore elettrospray rispetto all&#39;altro e l&#39;orifizio dello spettrometro di massa per raggiungere la massima intensità di ioni. </li><li> Selezionare la cella di interesse per l&#39;analisi utilizzando la vista dall&#39;alto sistema di visualizzazione. Dopo la selezione, spostare il palco per portare il campione cella selezionata direttamente sotto la punta affilata della fibra ottica. Utilizzando la vista laterale microscopio regolare nuovamente la punta-cellula distanza della superficie di circa 20 micron. </li><li> Impulsi laser fuoco alla cella selezionata attraverso la punta fibra inciso. Questo risultato nella perforazione della parete cellulare e l&#39;espulsione del citoplasma in forma di particolato. Dopo postionization dal electrospray, gli ioni prodotti sono raccolti dallo spettrometro di massa e gli spettri di massa sono registrati. </li><li> Dopo l&#39;acquisizione dei dati, mettere il laser, il electrospray e lo spettrometro di massa in modalità standby o disattivarli. Mantenere la A. ablazione cepa campione di tessuto per l&#39;osservazione al microscopio ottico opzionale. </li></ol> 6. Rappresentante Risultati Ablazione riuscita di un risultato singola cella nel scoppio della parete cellulare e la raccolta di uno spettro di massa LAESI. Un esperimento fallito risultati generalmente in nessun ablazione e / o non spettro di massa. A scopo dimostrativo, presentiamo i risultati del LAESI-MS analisi di una singola cellula epidermica incorporato di una A. CEPA lampadina. Figura 2a mostra un&#39;immagine rappresentativa di microscopia ottica, dopo il prelievo di una cellula. La parete cellulare è forata e l&#39;estensione della perforazione è limitata dai confini con le cellule vicine. Le celle adiacenti sembra essere intatto. Figura 2b mostra un rappresentante spettro di massa LAESI prodotta dalla cella. Una grande varietà di ioni vengono rilevati dalla A. cepa delle cellule e in base alle loro masse accurate, modelli di distribuzione isotopica e, in alcuni casi, spettri di massa tandem, sono provvisoriamente assegnati a metaboliti endogeni. Gli ioni rilevato da una singola cellula erano simili a quelli osservati per l&#39;analisi del tessuto stesso convenzionali LAESI-MS di più celle 13. <img src="/files/ftp_upload/2144/2144fig1.jpg" alt="Figura 1" /> Figura 1. Schematico di analisi singola cellula da LAESI-MS. Un mid-IR impulso laser è accoppiato ad una fibra ottica e consegnato al campione immessi sul vetrino. La punta della fibra affilato concentra la luce nella cella di destinazione e l&#39;energia depositata scoppia la cellula. Il pennacchio ablazione prodotti (punti rossi) è fusa con un pennacchio elettrospray (punti neri), le goccioline si uniscono (punti verdi) con conseguente produzione di ioni campione specifico. Questi ioni vengono analizzati e rilevati dal spettrometro di massa (MS). Due lunghi microscopi video di distanza, Microscopio Microscopio 1 e 2, sono utilizzati per monitorare distanza tra la punta della fibra e la superficie cellulare e selezionare una cella per l&#39;analisi, rispettivamente. <img src="/files/ftp_upload/2144/2144fig2a.jpg" alt="Figura 2a" /> Figura 2a. Ablazione segno su una singola cellula epidermica di una A. CEPA lampadina. Le celle adiacenti non presentano danni visibili e, successivamente, possono essere analizzati separatamente. <img src="/files/ftp_upload/2144/2144fig2b.jpg" alt="Figura 2b" /> Figura 2b. Spettro di massa corrispondente alla ablazione di un singolo A. cepa cellule epidermiche mostra la presenza di metaboliti primari e secondari.

<ol>
	<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><li>Chen, Z. 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=Early+plume+expansion+in+atmospheric+pressure+midinfrared+laser+ablation+of+water-rich+targets.">Early plume expansion in atmospheric pressure midinfrared laser ablation of water-rich targets.</a> Physical Review E. E77, 036316-036316 (2008).</li><li>Li, Y., 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=Atmospheric+Pressure+Molecular+Imaging+by+Infrared+MALDI.">Atmospheric Pressure Molecular Imaging by Infrared MALDI.</a> Mass Spectrometry. Analytical Chemistry. 79, 523-532 (2006).</li><li>Li, Y., 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=Atmospheric+Pressure+Infrared+MALDI+Imaging+Mass+Spectrometry+for+Plant+Metabolomics.">Atmospheric Pressure Infrared MALDI Imaging Mass Spectrometry for Plant Metabolomics.</a> Analytical Chemistry. 80, 407-420 (2007).</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> Analytical Chemistry. 79, 8098-8106 (2007).</li><li>Shrestha, B., 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=Rapid+analysis+of+pharmaceuticals+and+excreted+xenobiotic+and+endogenous+metabolites+with+atmospheric+pressure+infrared+MALDI+mass+spectrometry.">Rapid analysis of pharmaceuticals and excreted xenobiotic and endogenous metabolites with atmospheric pressure infrared MALDI mass spectrometry.</a> Metabolomics. 4, 297-311 (2008).</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> Analytical Chemistry. 80, 4575-4582 (2008).</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> Analytical Chemistry. 81, 6668-6675 (2009).</li><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> Analytical Chemistry. 82, 982-988 (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. , (2010).</li><li>Sripadi, P., Nazarian, J., Hathout, Y., Hoffman, E., 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+vitro+analysis+of+metabolites+from+the+untreated+tissue+of+Torpedo+californica+electric+organ+by+mid-infrared+laser+ablation+electrospray+ionization+mass+spectrometry.">In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry.</a> Metabolomics. 5, 263-276 (2009).</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> Analytical Chemistry. 81, 8265-8271 (2009).</li><li>Shrestha, B., 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=Ablation+and+Analysis+of+a+Single+Cell+by+Consecutive+Laser+Pulses.">Ablation and Analysis of a Single Cell by Consecutive Laser Pulses.</a> Applied Physics A. , (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

Il contenuto di acqua del citoplasma cellulare e la resistenza alla trazione della parete cellulare o la membrana sono i fattori critici che disciplinano l&#39;ablazione di singole cellule durante LAESI-MS. La fluenza laser deve essere regolata per raggiungere le soglie di ablazione dei diversi tipi di cellule. La fluenza laser consegnato dipende dall&#39;energia dell&#39;impulso laser e della distanza tra la punta della fibra e la superficie cellulare. Minimizzare la perturbazione delle cellule vicine durante l&#39;analisi singola cella è fondamentale. Mantenere l&#39;energia laser sulla soglia di ablazione limita l&#39;effetto di analisi sulle cellule adiacenti. Una delle potenziali applicazioni è usando le cellule unico come il naturale pixel volumetrico (voxel) per l&#39;industria chimica di imaging da MS. Rispetto alla raccolta di dati su una griglia rettangolare spesso artificiale, l&#39;imaging chimica di una cellula del tessuto, uno alla volta ha più probabilità di conservare l&#39;organizzazione spaziale delle interazioni biochimiche tra le cellule.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>45727</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>65548</td>
<td>&nbsp;</td>
<tr>
<td>1-methyl-2-pyrrolidinon</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>M79603</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>39259</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

direct analysis of single cells by mass spectrometry at atmospheric pressure

Analisi di sostanze biochimiche in singole cellule è importante per comprendere il metabolismo cellulare, ciclo cellulare, l&#39;adattamento, stati di malattia, ecc Anche i tipi di cellule stessa mostra eterogenea composizione biochimica a seconda delle loro condizioni fisiologiche e le interazioni con l&#39;ambiente. Metodi convenzionali di spettrometria di massa (MS) per l&#39;analisi di biomolecole in singole cellule contare su di preparazione del campione esteso. Rimuovere le cellule dal loro ambiente naturale e trattamento del campione ampio potrebbe portare a cambiamenti nella composizione cellulare. Metodi di ionizzazione ambiente consentire l&#39;analisi di campioni nel loro ambiente nativo e senza preparazione del campione esteso. 1 Le tecniche basate sul medio infrarosso (mid-IR) ablazione laser di materiali biologici a 2,94 micron di lunghezza d&#39;onda utilizzano l&#39;eccitazione improvvisa di acqua che si traduce in fase di esplosione. 2 tecniche di ionizzazione ambientale sulla base di mid-IR radiazione laser, come la ionizzazione elettrospray ablazione laser (LAESI) e pressione atmosferica infrarossi matrix-assisted laser ionizzazione desorbimento (IR AP-MALDI), hanno dimostrato con successo la capacità di analizzare direttamente acqua tessuti ricchi e biofluidi a pressione atmosferica. 3-11 LAESI Nella metà degli IR pennacchio ablazione laser che consiste principalmente di particelle neutre sotto il campione si fonde con le goccioline elettrospray alta carica per la produzione di ioni. Recentemente, a metà IR ablazione di singole cellule è stata effettuata con la consegna dei mid-IR radiazione attraverso una fibra inciso. Il pennacchio generato da questo ablazione è stata postionized da un elettrospray consentendo l&#39;analisi dei metaboliti diversi in singole cellule da LAESI-MS. 12 L&#39;articolo descrive il protocollo dettagliato per l&#39;analisi singola cella utilizzando LAESI-MS. Il video presentato mostra l&#39;analisi di una singola cellula epidermica dalla pelle di una lampadina Allium cepa. Lo schema del sistema è mostrata in Figura 1. Un esempio rappresentativo di ablazione singola cellula e spettro di massa LAESI dalla cella sono forniti in Figura 2.

Watch this Scientific Journal Video about Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure at JoVE.com

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analisi di singola cellula viene effettuata mediante spettrometria di massa su cellule animali e vegetali a pressione atmosferica, utilizzando una fibra ottica affilato per assaggiare le cellule per la ionizzazione elettrospray ablazione laser (LAESI) spettrometria di massa.

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 ...

direct-analysis-single-cells-mass-spectrometry-at-atmospheric

Research

JoVE Journal

Biology

15.9K Views.  George Washington University. Analisi di singola cellula viene effettuata mediante spettrometria di massa su cellule animali e vegetali a pressione atmosferica, utilizzando una fibra ottica affilato per assaggiare le cellule per la ionizzazione elettrospray ablazione laser (LAESI) spettrometria di massa.

Video: Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

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. ...

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

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

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 ...

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 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

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 ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

Analisi di LINE-1 retrotrasposizione al Nucleo livello singolo

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

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 ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

Preparazione del campione per l'analisi di proteomica massa-spettrometria-basato di microvasi oculari

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...