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: Este protocolo é desenvolvido para reduzir a heterogeneidade espacial do fragmento maltotriose e bradicinina (1-7) preparado com o método da gota seco. O protocolo consiste em três etapas principais, incluindo a preparação e pré-condicionamento, a deposição de amostra e de secagem e análise de dados de espectrometria de massa. Os procedimentos são descritos e descritos com mais detalhe a seguir: 1. Preparação e pré-condicionamento <ol><li> Limpeza da placa de amostra <ol><li> Usar luvas de borracha nitrílica e mão-lavar a placa de amostra suavemente com detergente e destilada-água deionizada (DDW). </li><li> Lavar a placa de amostra com metanol (MeOH) e DDW. </li><li> Insira a placa de amostra num copo de 600 ml e encher com DDW. </li><li> Sonicar a amostra na placa de DDW durante 15 minutos num banho de ultra-sons (200 W, 40 kHz). </li><li> Remover DDW a partir do recipiente e encher a proveta com MeOH. </li><li> Sonicar a placa de amostra em MeOH durante 15 min no banho de ultra-sons (200 W, 40 kHz). </li><li> Blow off as gotas de solvente na placa com azoto gasoso e manter a placa de amostra seca, antes da deposição da amostra. </li></ol></li><li> Regulação da temperatura de secagem Câmara NOTA:. A câmara de secagem é de 35 x 20 x 45 cm3 (W x D x H) câmara de acrílico Figura 1 mostra a imagem deste sistema de secagem. A câmara é purgado com gás azoto a temperatura ambiente através de um medidor de fluxo de gás a uma velocidade de fluxo constante para manter uma condição de baixa humidade relativa controlada por um higrómetro calibrado instalado no interior da câmara de secagem. Um bloco de base de cobre na câmara de secagem equipada com uma constante circulação de água de temperatura programado é utilizado para acomodar placas de amostra de aço inoxidável. O bloco de base de cobre é capaz de regular a temperatura da placa de amostra de 5 a 25 ° C. As temperaturas do ar, do bloco da base de cobre, e a placa de amostra são monitoradas por termopares tipo K. <ol><li> Abra a porta e colocar rapidamente a placa de amostra sobre o cobrebloco da base, em seguida, fechar a porta. </li><li> ajustar manualmente o medidor de vazão de gás para definir a taxa de fluxo de azoto a 10 pés cúbicos padrão por hora (scfh). </li><li> Monitorar a umidade relativa do ar na câmara de secagem pelo higrômetro e afinar o medidor de vazão de gás para garantir a umidade relativa do ar é sempre inferior a 25%. </li><li> Monitorar a temperatura da placa amostra por termopares tipo K e ajustar a temperatura da água de circulação manualmente até que o prato da amostra atinge 5 ° C durante experimento ou à temperatura ambiente (25 ° C) para o controlo. NOTA: De modo a estabilizar a placa de amostra a uma temperatura concebido, a temperatura de circulação de água é tipicamente definido de 0 a 5 ° C mais baixa do que a amostra concebido. Por exemplo, para manter a 5 ° C na placa de amostra, o ajuste da temperatura da bomba de circulação de água está compreendida no intervalo de 0 a 2 ° C; para manter a placa de amostras a 25 ° C, a regulação da temperatura da bomba de circulação de água é na gama de 23 a 25 ° C. </li><li> Assegurar que as temperaturas exigidas e a humidade relativa são atingidos (Tabela 1), antes da deposição da amostra. NOTA: Todos os parâmetros, bem como os seus valores de ajuste para os processos de secagem com temperaturas de chapa de amostra diferentes estão apresentados na Tabela 1. NOTA: a uma temperatura baixa placa de amostra, a condensação de água na chapa de amostra, pode ocorrer se a porta da câmara é aberta para um longo período de tempo. Se ocorrer condensação de água, fechar a porta e não depositar qualquer amostra nele até a condensação da água é seco. </li></ol></li><li> Preparação da matriz e do analito Soluções <ol><li> Preparação de soluções de matriz <ol><li> Preparar solução 0,1 M THAP com 50% de acetonitrilo (ACN): 50% solução aquosa de DDW. </li></ol></li><li> Preparação de analitos <ol><li> Prepare 10 -4 solução maltotriose M com DDW. </li><li> Preparar 10 -5 M fragmento bradicinina (1-7) em solução a 50% de acetonitrilo(ACN): 50% solução aquosa de DDW. </li></ol></li></ol></li></ol> 2. A deposição da amostra e Secagem <ol><li> Pré-mistura de 0,25 ml de solução 0,1 M THAP e 0,25 ul de 10 -4 M de maltotriose ou soluções em um tubo de microcentrífuga de 10 -5 M fragmento bradicinina (1-7). </li><li> Vortex a solução misturada por 3 s. </li><li> Centrifugar a solução mista de 2 seg (2000 xg) para recolher a solução no fundo do tubo de centrifugação. </li><li> Abrir a porta da câmara de secagem, cuidadosamente depositar 0,1 ul da solução na placa de amostra com uma pipeta e fechar a porta imediatamente. </li><li> Aguarde até que a gota de amostra a secar. . NOTA: Os tempos de secagem tipicamente observada com temperaturas de chapa de amostra diferentes estão listados na Tabela 1 para a temperatura da placa de amostra de 5 ° C, o tempo médio de secagem é de 800 a 1000 seg; para temperatura da placa de amostra de 25 ° C, o tempo médio de secagem é de 100 a 150 sec. </li><li> Após a secagem, abrir a porta da câmara de secagem. </li><li> Ajustar a temperatura da água de circulação até à temperatura ambiente (25 ° C). NOTA: Ignore este passo se a placa de amostra é mantida constantemente à temperatura ambiente (25 ° C) durante o processo de secagem. </li><li> Depois da amostra de temperatura da placa de voltar à temperatura ambiente (25 ° C), remover o prato da amostra a partir da câmara de secagem. </li><li> Examinar a morfologia da amostra sob um microscópio estereoscópico de 5x e tomar imagem de campo brilhante um instantâneo. NOTA: Se as morfologias de cristal não são como esperado, é necessário preparar uma nova amostra com o mesmo procedimento. Morfologias cristalinas típicas são apresentadas nos painéis superior da Figura 2. NOTA: Nos casos com temperaturas de chapa de amostra baixa, tal como 5 ° C, é importante para aquecer a placa de amostra para a temperatura ambiente antes de o levar para fora da câmara de secagem. Ao depositar as amostras, não mantenha o solutio pré-misturadon na ponta da pipeta ao longo de 10 seg. NÃO usar a solução pré-misturada de novo depois de depositar as amostras. Os painéis superior da Figura 2 mostrar as imagens de campo brilhante de amostras preparadas com temperaturas de chapa de amostra diferentes. </li></ol> Análise 3. Espectrometria de Massa de dados <ol><li> Dados de espectrometria de massa Aquisição NOTA: Após a preparação, a amostra pode ser analisada utilizando espectrometria de massa de imagem. No presente estudo, as experiências de imagiologia MS são realizados utilizando um sincronizado TOF dupla polaridade construído em laboratório (DP-TOF) espectrómetro de massa de imagiologia. 15 espectrómetros de massa MALDI-TOF comercial com capacidade de imagiologia também são adequados para tais experiências. O espectrómetro de massa é operado na extração linear e modos de iões positivos com atrasos de extração otimizados. A energia cinética dos iões é de 20 kV. O tamanho do feixe de laser é de 35 um de diâmetro sobre a superfície da amostra, e o espectro de cada ponto representa a avraiva de 5 disparos de laser. <ol><li> Insira a placa de amostra para o espectrómetro de massa MALDI. </li><li> Realizar imagem análise de espectrometria de massa para a amostra preparada em passos 2.1-2.9. </li><li> Selecione um pico de massa característica da lista de massa mostrado na janela de resultados e clique em &quot;2D&quot; para traçar uma imagem ion bidimensional. NOTA: Para maltotriose misturado com THAP, os picos característicos são sodiated maltotriose, THAP protonada e sodiated THAP. Por fragmento de bradicinina (1-7) misturado com THAP, os picos característicos incluem protonado fragmento bradicinina (1-7), THAP protonado, e sodiated THAP. </li><li> Clique nos botões de ajuste na janela de pop-up para determinar os limites superior e inferior da intensidade do sinal e clique em &quot;salvar uma imagem&quot;. Esta configuração define o contraste das imagens íon. NOTA: Em cada conjunto individual de dados, as regiões rachados e os pontos nulos mostrando baixa luminosidade são eliminados. </li><li> Observar e comparar o íonimagem com a imagem de campo brilhante que foi tomada no passo 2.9. NOTA: Geração de espectrometria de massa e produção de imagens de iões específicos pode ser conseguida com instrumentos comerciais. Devido à variedade de aquisição de dados e software de análise, os usuários devem seguir as instruções de software fornecidas pelo fornecedor do instrumento para obter imagens de alta qualidade. </li></ol></li><li> Análise de dados NOTA: A heterogeneidade das amostras é analisado quantitativamente. Nesta demonstração, cada amostra é dividida em várias áreas concêntricas por software desenvolvido in-house para analisar a distribuição espacial dos íons. A análise pode também ser realizada usando software de análise de dados independente. <ol><li> Clique os pontos nulos e as regiões rachados na imagem ion mostrado na janela de resultado para remover as áreas sem importância. NOTA: Este procedimento define a área essencial imagem de iões de. </li><li> Clique em &quot;encontrar edge&quot; botão para encontrar a camada mais externa da imagem de ion. </li><li> Clique em &quot;deduzir&quot; para salvar as informações abundância de íons da camada mais externa em um banco de dados e remover esta camada a partir da imagem de íon simultaneamente. Uma caixa de seleção que representa esta camada mais externa aparecerá na lista &quot;de dados de saída&quot; da janela de resultados. </li><li> Repita os passos 3.2.2 e 3.2.3 até que o centro da imagem de iões é definido. </li><li> Clique e selecione todas as caixas de seleção na lista de &quot;dados de saída&quot; e clique em &quot;Exportar&quot; para exportar os dados. </li><li> Abrir os dados exportados usando o software de planilha para calcular a abundância média de iões de cada camada para obter as informações de distribuição espacial de íons. </li></ol></li></ol>

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The authors declare no competing financial interest.

Com base em previsões teóricas anteriores, fluxos hidrodinâmicos induzidas pela temperatura dentro de gotículas pode ultrapassar para fora fluxos capilares induzidas por evaporação do solvente. A eficiência de tais moléculas de recirculação interna é aumentada quando a gradientes de temperatura dentro de um aumento de gotícula. De acordo com os resultados previstos, ao manter a temperatura da placa de amostra abaixo de 5 ° C, mantendo a sua envolvente, à temperatura ambiente, a velocidade média de fluxo d...

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">
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		<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.

As imagens de campo brilhante, bem como as imagens de MS fragmento maltotriose e bradicinina (1-7) preparado, com a temperatura da placa de amostra de 5 e 25 ° C são apresentados na Figura 1. No caso de maltotriose sodiated, o sinal de iões principalmente povoa na periferia da área de amostra quando é preparado com uma temperatura de placa de amostra de 25 ° C. Diminuindo a temperatura da placa de amostra a 5 ° C, o sinal preenche de forma homogénea sobre toda a ...

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

Um protocolo para reduzir as heterogeneidades espaciais dos sinais de iões em espectrometria de massa MALDI por regulação da temperatura do substrato durante o processo de secagem da amostra é demonstrada.

Preparação de Amostras de MALDI homogênea para Aplicações quantitativas

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 visualizações.  Academia Sinica.  Um protocolo para reduzir as heterogeneidades espaciais dos sinais de iões em espectrometria de massa MALDI por regulação da temperatura do substrato durante o processo de secagem da amostra é demonstrada. 

Vídeo: 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.

O isolamento de lipoproteínas de alta densidade para não codificante pequeno ARN Quantificação

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

Bioquímica

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.

Sheathless Eletroforese Capilar-Espectrometria de Massa para Metabolic Profiling de amostras biológicas

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.

Fácil manipulação das arquiteturas em hidrogel à base de proteínas para aplicações de cultura de células

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.

Preparação ideal de formol fixada amostras para peptídeo baseado Matrix assistida por espectrometria de massa de ionização/dessorção Laser Imaging fluxos de trabalho

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.

Revisão e ensaio de reparo de DNA usando extensão de nucleotídeo único e análise de espectrometria de massa 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 de Termoforese microescala para medir interações proteína-lipídicas

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.

Análise Quantitativa do Lipidome Celular de Saccharomyces Cerevisiae Usando Cromatografia Líquida Juntamente com Espectrometria de Massa de 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.

Análise da cadeia de Ubiquitin por monitoramento de reação paralela

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.

Metabolômica Quantitativa de Saccharomyces Cerevisiae Usando cromatografia líquida juntamente com espectrometria de 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.

Imagem de molécula única de condensados EWS-FLI1 montados no 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 ...