This work is supported by the Genomics Research Center, Academia Sinica and the Ministry of Science and Technology of Taiwan, the Republic of China (Contract No. 104-2119-M-001-014).

NOTA: Questo protocollo è sviluppato per ridurre l&#39;eterogeneità spaziale maltotrioso e bradichinina frammento (1-7) preparato con il metodo essiccati gocciolina. Il protocollo consiste di tre fasi principali, compresa la preparazione e precondizionamento, la deposizione del campione e l&#39;essiccazione, e l&#39;analisi dei dati di spettrometria di massa. Le procedure sono delineati e descritti più in dettaglio qui di seguito: 1. Preparazione e precondizionamento <ol><li> Pulizia del piatto del campione <ol><li> Indossare guanti di nitrile e mano-lavare delicatamente la piastra campione con detergente e acqua distillata-deionizzata (DDW). </li><li> Lavare il piatto del campione con metanolo (MeOH) e DDW. </li><li> Inserire la piastra campione in un becher da ml 600 e riempire con DDW. </li><li> Sonicare piatto del campione in DDW per 15 minuti in un bagno a ultrasuoni (200 W, 40 kHz). </li><li> Rimuovere DDW dal bicchiere e riempire il bicchiere con MeOH. </li><li> Sonicare piatto del campione in MeOH per 15 minuti in bagno ad ultrasuoni (200 W, 40 kHz). </li><li> Soffiare via le gocce di solvente sul piatto con gas azoto e mantenere la piastra campione asciutto prima deposizione del campione. </li></ol></li><li> Regolazione della temperatura di asciugatura Camera NOTA:. La camera di essiccazione è un 35 x 20 x 45 cm 3 (L x P x A) camera di acrilico La figura 1 mostra l&#39;immagine di questo sistema di asciugatura. La camera è pulita con temperatura ambiente azoto gassoso attraverso un misuratore di flusso di gas ad una portata costante per mantenere una condizione di bassa umidità relativa monitorata da un igrometro calibrato installato all&#39;interno della camera di essiccazione. Un blocco base di rame nella camera di essiccazione dotato di una costante circolatore dell&#39;acqua temperatura programmata Serve per targhe di esempio in acciaio inox. Il blocco base in rame è in grado di regolare la temperatura della piastra del campione da 5 a 25 ° C. Le temperature dell&#39;aria, blocco base di rame, e la piastra sono controllate da K-termocoppie. <ol><li> Aprire la porta e rapidamente mettere piatto del campione sul rameblocco di base quindi chiudere la porta. </li><li> Regolare manualmente il misuratore di portata di gas per impostare il flusso di azoto a 10 piedi cubi standard per ora (SCFH). </li><li> Monitorare l&#39;umidità relativa nella camera di essiccazione dal igrometro e calibrare il misuratore di portata di gas per assicurare l&#39;umidità relativa è sempre inferiore al 25%. </li><li> Monitorare la temperatura del piatto del campione mediante termocoppie di tipo K e regolare manualmente la temperatura dell&#39;acqua circolatore finché la piastra campione raggiunge 5 ° C per esperimento oa temperatura ambiente (25 ° C) per il controllo. NOTA: Al fine di stabilizzare il piatto del campione ad una temperatura progettato, la temperatura dell&#39;acqua circolatore è in genere impostato tra 0 e 5 ° C inferiore rispetto al campione progettato. Ad esempio, per mantenere 5 ° C al piatto del campione, la temperatura del circolatore acqua è nell&#39;intervallo da 0 a 2 ° C; per mantenere la piastra di campione a 25 ° C, la temperatura del circolatore acqua è nell&#39;intervallo da 23 a 25 ° C. </li><li> Assicurarsi che le temperature richieste e l&#39;umidità relativa vengono raggiunti (Tabella 1) prima della deposizione del campione. NOTA: Tutti i parametri ei loro valori di regolazione per i processi di essiccazione a temperature diverse piastre campione sono riportati in Tabella 1. NOTA: A bassa temperatura piatto del campione, condensa sulla piastra campione può verificarsi se la porta della camera aperta per lungo tempo. In caso di condensa di acqua, chiudere la porta e NON depositare qualsiasi campione su di esso fino a quando la formazione di condensa è asciugata. </li></ol></li><li> Preparazione di Matrix e Analita Solutions <ol><li> Preparazione delle soluzioni matrice <ol><li> Preparare 0,1 soluzione M THAP con il 50% acetonitrile (ACN): 50% soluzione acquosa DDW. </li></ol></li><li> Preparazione di analiti <ol><li> Preparare 10 -4 soluzione maltotriosio M con DDW. </li><li> Preparare 10 -5 M frammento bradichinina (1-7) soluzione nel 50% acetonitrile(ACS): 50% soluzione acquosa DDW. </li></ol></li></ol></li></ol> 2. Deposizione del campione e asciugatura <ol><li> Premix 0,25 ml di soluzione di THAP 0,1 M e 0,25 ml di 10 -4 M maltotriosio o 10 -5 M frammento bradichinina (1-7) soluzioni in una provetta. </li><li> Agitare la soluzione mista per 3 sec. </li><li> Centrifugare la soluzione mista per 2 sec (2.000 xg) per raccogliere la soluzione al fondo della provetta. </li><li> Aprire la porta della camera di essiccazione, accuratamente depositare 0,1 ml di soluzione sul piatto del campione con pipetta e chiudere immediatamente la porta. </li><li> Attendere che la goccia del campione si asciughi. . NOTA: I tempi di essiccazione tipicamente osservati con differenti temperature piatto del campione sono elencati nella tabella 1 per la temperatura della piastra campione di 5 ° C, il tempo medio di asciugatura è di 800 a 1.000 sec; per temperatura della piastra campione di 25 ° C, il tempo medio di essiccazione è da 100 a 150 sec. </li><li> Dopo l&#39;essiccazione, aprire la porta della camera di essiccazione. </li><li> Impostare la temperatura dell&#39;acqua circolatore a temperatura ambiente (25 ° C). NOTA: Saltare questo punto se la piastra campione viene mantenuta costantemente a temperatura ambiente (25 ° C) durante il processo di essiccazione. </li><li> Dopo la temperatura della piastra campione ritorni a temperatura ambiente (25 ° C), rimuovere la piastra campione dalla camera di essiccazione. </li><li> Esaminare la morfologia del campione allo stereomicroscopio 5X e prendere l&#39;immagine in campo chiaro un&#39;istantanea. NOTA: Se le morfologie cristalline non sono come previsto, è necessario preparare un nuovo campione con la stessa procedura. Morfologie cristalline tipiche sono illustrate nei pannelli superiori della figura 2. NOTA: Nei casi con temperature piastra campione basse, ad esempio 5 ° C, è importante riscaldare il piatto del campione a temperatura ambiente, mette fuori dalla camera di essiccazione. All&#39;atto del deposito dei campioni, non mantenere la solutio premiscelaton nella punta della pipetta di oltre 10 secondi. NON usare di nuovo la soluzione premiscelata dopo aver depositato i campioni. I pannelli superiori della figura 2 mostrano immagini in campo chiaro di campioni preparati con diverse temperature della piastra del campione. </li></ol> Analisi 3. Spettrometria di massa dei dati <ol><li> Spettrometria di Massa Data Acquisition NOTA: Dopo la preparazione, il campione può essere analizzato utilizzando la spettrometria di massa imaging. Nel corso di studio, gli esperimenti di imaging MS sono condotti utilizzando un sincronizzato TOF doppia polarità laboratorio integrato (DP-TOF) spettrometro di massa di imaging. 15 Commercial MALDI-TOF spettrometri di massa con capacità di imaging sono adatti a tali esperimenti anche. Lo spettrometro di massa è gestito in estrazione lineare e modalità di ioni positivi con ritardi di estrazione ottimizzate. L&#39;energia cinetica degli ioni è di 20 kV. La dimensione del fascio laser è di 35 micron di diametro sulla superficie del campione, e lo spettro di ogni punto è la avela rabbia di 5 colpi laser. <ol><li> Inserire la piastra del campione nello spettrometro di massa MALDI. </li><li> Eseguire l&#39;imaging spettrometria di massa per il campione preparato nei passaggi 2.1-2.9. </li><li> Selezionare un picco di massa caratteristica dall&#39;elenco di massa mostrato nella finestra dei risultati e fare clic su &quot;2D&quot; per tracciare un&#39;immagine ioni bidimensionale. NOTA: Per maltotriosio mescolato con THAP, i picchi caratteristici sono sodiated maltotriosio, THAP protonata e sodiated THAP. Per frammento bradichinina (1-7) miscelato con THAP, i picchi caratteristici comprendono protonati frammento bradichinina (1-7), protonata THAP, e sodiated THAP. </li><li> Fare clic sui pulsanti di regolazione nella finestra pop-up per determinare i limiti superiore e inferiore della intensità del segnale e fare clic su &quot;salvare un&#39;immagine&quot;. Questa impostazione definisce il contrasto delle immagini di ioni. NOTA: in ogni set individuale dei dati, le regioni e le macchie screpolate nulli mostrano bassa luminosità sono eliminati. </li><li> Osservare e confrontare lo ioneimmagine l&#39;immagine in campo chiaro che è stata presa al punto 2.9 con. NOTA: imaging con spettrometria di massa e costruzione di immagini di particolari ioni può essere realizzato con strumenti commerciali. A causa della varietà di acquisizione dati e software di analisi, gli utenti devono seguire le istruzioni software forniti dal fornitore dello strumento per ottenere immagini di alta qualità. </li></ol></li><li> Analisi dei dati NOTA: L&#39;eterogeneità dei campioni viene analizzato quantitativamente. In questa dimostrazione, ogni campione viene diviso in più zone concentriche di software sviluppati internamente per analizzare la distribuzione spaziale degli ioni. L&#39;analisi può essere eseguita anche utilizzando software di analisi dati stand-alone. <ol><li> Clicca i punti nulli e le regioni incrinate l&#39;immagine di ioni mostrata nella finestra dei risultati in per rimuovere aree non importanti. NOTA: Questa procedura definisce l&#39;area essenziale dell&#39;immagine ionico. </li><li> Fare clic sul pulsante &quot;edge trovare&quot; per trovare strato esterno dell&#39;immagine di ioni. </li><li> Fai clic su &quot;dedurre&quot; per salvare le informazioni ioni abbondanza dello strato esterno in un database e rimuovere questo strato dall&#39;immagine ioni contemporaneamente. Una casella di controllo che rappresenta questo strato più esterno apparirà nel &quot;dati di uscita&quot; lista della finestra dei risultati. </li><li> Ripetere i punti 3.2.2 e 3.2.3 fino viene definito il centro dell&#39;immagine ione. </li><li> Fare clic e selezionare tutte le caselle di controllo nella lista &quot;dati di uscita&quot; e fare clic su &quot;Esporta&quot; per esportare i dati. </li><li> Aprire i dati esportati utilizzando il software foglio di calcolo per calcolare l&#39;abbondanza media di ioni di ogni livello per ottenere le informazioni distribuzione spaziale degli ioni. </li></ol></li></ol>

<ol>
	<li>Karas, M., Hillenkamp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Desorption+Ionization+of+Proteins+with+Molecular+Masses+Exceeding+10000+Daltons.">Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons.</a> Anal. Chem. 60, 2299-2301 (1988).</li><li>Beavis, R. C., 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=Velocity+Distributions+of+Intact+High+Mass+Polypeptide+Molecule+Ions+Produced+by+Matrix+Assisted+Laser+Desorption.">Velocity Distributions of Intact High Mass Polypeptide Molecule Ions Produced by Matrix Assisted Laser Desorption.</a> Chem. Phys. Lett. 181, 479-484 (1991).</li><li>Beavis, R. C., Chaudhary, T., 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=Alpha-Cyano-4-Hydroxycinnamic+Acid+as+a+Matrix+for+Matrix-Assisted+Laser+Desorption+Mass-Spectrometry.">Alpha-Cyano-4-Hydroxycinnamic Acid as a Matrix for Matrix-Assisted Laser Desorption Mass-Spectrometry.</a> Org. Mass Spectrom. 27, 156-158 (1992).</li><li>Ehring, H., Karas, M., Hillenkamp, F. <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+Photoionization+and+Photochemistry+in+Ionization+Processes+of+Organic-Molecules+and+Relevance+for+Matrix-Assisted+Laser+Desorption+Ionization+Mass-Spectrometry.">Role of Photoionization and Photochemistry in Ionization Processes of Organic-Molecules and Relevance for Matrix-Assisted Laser Desorption Ionization Mass-Spectrometry.</a> Org. Mass Spectrom. 27, 472-480 (1992).</li><li>Strupat, K., Karas, M., Hillenkamp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,5-Dihydroxybenzoic+Acid+-+a+New+Matrix+for+Laser+Desorption+Ionization+Mass-Spectrometry.">2,5-Dihydroxybenzoic Acid - a New Matrix for Laser Desorption Ionization Mass-Spectrometry.</a> Int. J. Mass Spectrom. Ion Process. 111, 89-102 (1991).</li><li>Hu, H., Larson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaporation+of+a+Sessile+Droplet+on+a+Substrate.">Evaporation of a Sessile Droplet on a Substrate.</a> J. Phys. Chem. B. 106, 1334-1344 (2002).</li><li>Deegan, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Flow+as+the+Cause+of+Ring+Stains+from+Dried+Liquid+Drops.">Capillary Flow as the Cause of Ring Stains from Dried Liquid Drops.</a> Nature. 389, 827-829 (1997).</li><li>Hu, J. -. B., Chen, Y. -. C., Urban, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coffee-Ring+Effects+in+Laser+Desorption/Ionization+Mass+Spectrometry.">Coffee-Ring Effects in Laser Desorption/Ionization Mass Spectrometry.</a> Anal. Chim. Acta. 766, 77-82 (2013).</li><li>Schwartz, S. A., Reyzer, M. L., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Tissue+Analysis+Using+Matrix-Assisted+Laser+Desorption/Ionization+Mass+Spectrometry:+Practical+Aspects+of+Sample+Preparation.">Direct Tissue Analysis Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: Practical Aspects of Sample Preparation.</a> J. Mass Spectrom. 38, 699-708 (2003).</li><li>Hu, H., Larson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marangoni+Effect+Reverses+Coffee-Ring+Depositions.">Marangoni Effect Reverses Coffee-Ring Depositions.</a> J. Phys. Chem. B. 110, 7090-7094 (2006).</li><li>Bhardwaj, R., Fang, X., Attinger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+Formation+During+the+Evaporation+of+a+Colloidal+Nanoliter+Drop:+A+Numerical+and+Experimental+Study.">Pattern Formation During the Evaporation of a Colloidal Nanoliter Drop: A Numerical and Experimental Study.</a> New J. Phys. 11, 075020 (2009).</li><li>Savino, R., Paterna, D., Favaloro, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buoyancy+and+Marangoni+Effects+in+an+Evaporating+Drop.">Buoyancy and Marangoni Effects in an Evaporating Drop.</a> J Thermophys Heat Tr. 16, 562-574 (2002).</li><li>Probstein, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Surface Tension. in Physicochemical Hydrodynamics : An Introduction. , 305-361 (1994).</li><li>Lai, Y. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+Spatial+Heterogeneity+of+MALDI+Samples+with+Marangoni+Flows+During+Sample+Preparation.">Reducing Spatial Heterogeneity of MALDI Samples with Marangoni Flows During Sample Preparation.</a> J. Am. Soc. Mass Spectrom. 27, 1314-1321 (2016).</li><li>Hsiao, C. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Molecular+Imaging+of+Photolabile+Surface+Samples+with+Synchronized+Dual-Polarity+Time-of-Flight+Mass+Spectrometry.">Comprehensive Molecular Imaging of Photolabile Surface Samples with Synchronized Dual-Polarity Time-of-Flight Mass Spectrometry.</a> Rapid Commun. Mass Spectrom. 25, 834-842 (2011).</li><li>Vorm, O., Roepstorff, P., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Resolution+and+Very+High-Sensitivity+in+MALDI+TOF+of+Matrix+Surfaces+Made+by+Fast+Evaporation.">Improved Resolution and Very High-Sensitivity in MALDI TOF of Matrix Surfaces Made by Fast Evaporation.</a> Anal. Chem. 66, 3281-3287 (1994).</li><li>Gabriel, S. J., Schwarzinger, C., Schwarzinger, B., Panne, U., Weidner, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Segregation+as+the+Major+Cause+for+Sample+Inhomogeneity+in+MALDI+Dried+Droplet+Spots.">Matrix Segregation as the Major Cause for Sample Inhomogeneity in MALDI Dried Droplet Spots.</a> J. Am. Soc. Mass Spectrom. 25, 1356-1363 (2014).</li><li>Mampallil, D., Eral, H. B., van den Ende, D., Mugele, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Evaporating+Complex+Fluids+through+Electrowetting.">Control of Evaporating Complex Fluids through Electrowetting.</a> Soft Matter. 8, 10614-10617 (2012).</li></ol>

The authors declare no competing financial interest.

Sulla base di precedenti previsioni teoriche, temperatura indotta flussi idrodinamici all&#39;interno gocce possono superare verso l&#39;esterno flussi capillari indotte da evaporazione del solvente. L&#39;efficacia di tale ricircolo interno delle molecole è maggiore quando la temperatura gradienti di un aumento gocciolina. Secondo i risultati previsti, quando mantenendo la temperatura della piastra campione sotto 5 ° C, mantenendo l&#39;ambiente circostante a temperatura ambiente, la velocità media dei flussi di ric...

Mass spectrometry (MS) is one of the most important analytical techniques for analyzing the molecular compositions of complex samples. Among all the ionization methods used in MS, matrix-assisted laser desorption/ionization (MALDI) is the most sensitive and widely used method in bioanalytical applications.1 In comparison to other ionization techniques, MALDI has the highest sensitivity and high tolerance to salt contaminants. Such analytical properties make MALDI the first choice for carbohydrate analysis and many proteomics applications. However, sample preparation is a crucial step for obtaining high quality data in MALDI-MS.
The most commonly used sample preparation method for MALDI-MS is the dried-droplet method, in which sample droplets are deposited on a surface and dried under ambient conditions. This drying method is simple and generally effective.2-5 However, a common problem in the dried-droplet method is that the resultant analyte/matrix crystals normally distribute irregularly. In many cases, the crystals aggregate at the periphery of sample areas, resulting in the so-called ring-stain formation.6-8 The heterogeneous crystal morphologies affect the spatial distribution of analyte molecules, which results in severe fluctuation in ion signal over sample areas. Such severe signal fluctuations and poor data reproducibility are known as the "sweet spot" problem in MALDI-MS.9 Thus, there is a great need for reducing spatial heterogeneities in MALDI-MS dried droplet applications.
Hydrodynamic flows in the sample droplet play an important role in determining the spatial distribution of samples prepared with the dried-droplet method.10-12 It was found that the evaporation of solvent induces outward capillary flows within droplets, which are responsible for the ring-stain formation.7,10 In contrast, recirculation flows induced by tangential surface-tension gradients may counterbalance the outward capillary flows.13 If the recirculation flow speeds are higher than that of the outward capillary flows, the samples can be efficiently redistributed to reduce the heterogeneity problem.14
In this work, we demonstrate a detailed protocol for preparing samples with a simple drying chamber to induce efficient recirculation flows during droplet drying processes. Droplet drying conditions are precisely controlled, including the temperatures of the sample plate and its surroundings, and the relative humidity within the chamber. The model analytes include maltotriose and bradykinin chain (1-7). The matrix used for the demonstration is 2,4,6-trihydroxyacetophenone (THAP). The samples are examined with time-of-flight (TOF) MS, and the data are analyzed quantitatively to show the reduction of heterogeneity.

<table border="0" cellpadding="0" cellspacing="0" width="787">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Reagent</td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Detergent powder</td>
			<td style="width:157px;">Alconox</td>
			<td style="width:139px;">242985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Methanol</td>
			<td style="width:157px;">Merck</td>
			<td style="width:139px;">106009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Acetonitrile</td>
			<td style="width:157px;">Merck</td>
			<td style="width:139px;">100003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">2,4,6-trihydroxyacetophenone (THAP)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">T64602&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bradykinin fragment (1-7)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">B1651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Maltotriose</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">47884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette tips</td>
			<td style="width:157px;">Mettler Toledo</td>
			<td style="width:139px;">17005091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microcentrifuge tube</td>
			<td style="width:157px;">Axygen</td>
			<td style="width:139px;">MCT-150-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Milli-Q water purification system</td>
			<td style="width:157px;">Millipore</td>
			<td style="width:139px;">ZMQS6VFT1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powder-free nitrile gloves</td>
			<td style="width:157px;">Microflex</td>
			<td style="width:139px;">SU-690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">600 mL beaker</td>
			<td style="width:157px;">Duran</td>
			<td style="width:139px;">2110648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ultrasonic cleaner</td>
			<td style="width:157px;">Delta</td>
			<td style="width:139px;">DC300H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hygrometer</td>
			<td style="width:157px;">Wisewind</td>
			<td style="width:139px;">5330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nitrogen gas flowmeter</td>
			<td style="width:157px;">Dwyer</td>
			<td style="width:139px;">RMA-6-SSV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">K-type thermocouples</td>
			<td style="width:157px;">Digitron</td>
			<td style="width:139px;">311-1670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Centrifuge</td>
			<td style="width:157px;">Select BioProducts</td>
			<td style="width:139px;">Force Mini&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette</td>
			<td style="width:157px;">Rainin</td>
			<td style="width:139px;">pipet-lite XLS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Stereomicroscope</td>
			<td style="width:157px;">Olympus</td>
			<td style="width:139px;">SZX16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Temperature controllable drying chamber</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Synchronized dual-polarity time-of-flight imaging mass spectrometer (DP-TOF IMS)</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">MALDI-TOF stainless steel sample target</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of homogeneous maldi samples for quantitative applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

Le immagini in campo chiaro e le immagini MS di maltotrioso e bradichinina frammento (1-7) preparato con temperatura della piastra campione di 5 e 25 ° C sono mostrati in Figura 1. Nel caso di maltotriosio sodiated, il segnale ione principalmente popola alla periferia dell&#39;area campione quando viene preparato con una temperatura della piastra campione di 25 ° C. Diminuendo la temperatura della piastra del campione a 5 ° C, il segnale popola omogeneamente su tutta ...

Watch this Scientific Journal Video about Preparation of Homogeneous MALDI Samples for Quantitative Applications at JoVE.com

Preparation of Homogeneous MALDI Samples for Quantitative Applications

Lo dimostra un protocollo per la riduzione eterogeneità spaziali di segnali di ioni in spettrometria di massa MALDI regolando la temperatura del substrato durante i processi di essiccazione del campione.

Preparazione dei campioni MALDI omogenei per applicazioni quantitative

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

preparation-of-homogeneous-maldi-samples-for-quantitative-applications

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

8.7K Views.  Academia Sinica.  Lo dimostra un protocollo per la riduzione eterogeneità spaziali di segnali di ioni in spettrometria di massa MALDI regolando la temperatura del substrato durante i processi di essiccazione del campione. 

Video: Preparation of Homogeneous MALDI Samples for Quantitative Applications

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

Isolamento di lipoproteine ​​ad alta densità per non codificante Piccolo RNA Quantificazione

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Ricerca

Biochimica

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

Guaina elettroforesi capillare-spettrometria di massa per metaboliche Profiling di campioni biologici

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Facile manipolazione delle architetture in idrogel a base di proteine per applicazioni della coltura delle cellule

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

Preparazione ottimale di formalina riparata campioni per Peptide basati su matrice assistita spettrometria totale di desorbimento/ionizzazione Laser i flussi di lavoro di Imaging

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

Correzione di bozze e saggio di riparazione del DNA usando l'analisi di spettrometria di massa di singolo Nucleotide estensione e MALDI-TOF

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Uso della termoforesi su microscala per misurare le interazioni proteina-lipidi

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Analisi quantitativa del Lipidome cellulare di Saccharomyces Cerevisiae Utilizzando la cromatografia Liquida Accoppiata con la Spettrometria di Massa Tandem

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

Analisi della catena Ubiquitin mediante monitoraggio parallelo della reazione

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Metabolomica quantitativa di Saccharomyces Cerevisiae mediante cromatografia liquida accoppiata con spettrometria di massa tandem

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

Imaging a singola molecola dell'assemblaggio di condensati EWS-FLI1 sul DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Preparazione dei campioni MALDI omogenei per applicazioni quantitative

Riepilogo

Esplora Altri Video

Capitoli in questo video

Isolamento di lipoproteine ad alta densità per non codificante Piccolo RNA Quantificazione

Guaina elettroforesi capillare-spettrometria di massa per metaboliche Profiling di campioni biologici

Facile manipolazione delle architetture in idrogel a base di proteine per applicazioni della coltura delle cellule

Preparazione ottimale di formalina riparata campioni per Peptide basati su matrice assistita spettrometria totale di desorbimento/ionizzazione Laser i flussi di lavoro di Imaging

Correzione di bozze e saggio di riparazione del DNA usando l'analisi di spettrometria di massa di singolo Nucleotide estensione e MALDI-TOF

Uso della termoforesi su microscala per misurare le interazioni proteina-lipidi

Analisi quantitativa del Lipidome cellulare di Saccharomyces Cerevisiae Utilizzando la cromatografia Liquida Accoppiata con la Spettrometria di Massa Tandem

Analisi della catena Ubiquitin mediante monitoraggio parallelo della reazione

Metabolomica quantitativa di Saccharomyces Cerevisiae mediante cromatografia liquida accoppiata con spettrometria di massa tandem

Imaging a singola molecola dell'assemblaggio di condensati EWS-FLI1 sul DNA

Preparazione dei campioni MALDI omogenei per applicazioni quantitative

Riepilogo

Esplora Altri Video

Capitoli in questo video

Isolamento di lipoproteine ​​ad alta densità per non codificante Piccolo RNA Quantificazione

Guaina elettroforesi capillare-spettrometria di massa per metaboliche Profiling di campioni biologici

Facile manipolazione delle architetture in idrogel a base di proteine per applicazioni della coltura delle cellule

Preparazione ottimale di formalina riparata campioni per Peptide basati su matrice assistita spettrometria totale di desorbimento/ionizzazione Laser i flussi di lavoro di Imaging

Correzione di bozze e saggio di riparazione del DNA usando l'analisi di spettrometria di massa di singolo Nucleotide estensione e MALDI-TOF

Uso della termoforesi su microscala per misurare le interazioni proteina-lipidi

Analisi quantitativa del Lipidome cellulare di Saccharomyces Cerevisiae Utilizzando la cromatografia Liquida Accoppiata con la Spettrometria di Massa Tandem

Analisi della catena Ubiquitin mediante monitoraggio parallelo della reazione

Metabolomica quantitativa di Saccharomyces Cerevisiae mediante cromatografia liquida accoppiata con spettrometria di massa tandem

Imaging a singola molecola dell'assemblaggio di condensati EWS-FLI1 sul DNA

Isolamento di lipoproteine ad alta densità per non codificante Piccolo RNA Quantificazione