Este trabalho foi financiado pela National Science Foundation (MCB 1.021.831, JM) e J. &amp; E. Programa de Pesquisa Biomédica (Florida Department of Health, JM).

1. Preparações de amostras <ol><li> Realizar a preparação de amostras e todas as manipulações de amostra em um quarto escuro para evitar uma uncaging indesejada. </li><li> Solubiliza-DM-nitrophen ((1 - (2-nitro-4 ,5-dimetoxifenil) - N, N, N &#39;, N&#39;-tetraquis [(oxi-carbonil) metil] -1,2-etanodiamina) em HEPES 50 mM tampão, KCl 100 mM, pH 7,0 para uma concentração final de 400 uM (ε 350nm = 4330 M -1 cm -1 9). </li><li> Adicionar CaCl2 a partir da solução de estoque 0,1 M para atingir uma proporção desejável de [Ca 2 +]: [DM-nitrophen]. Para as proteínas com a K d para o Ca2 + associação maior do que 10 uM, a proporção de [Ca 2 +]: [DM-nitrophen] de 1:01 é preferível para evitar a ligação de Ca libertado foto-2 + para uncaged DM-nitrophen . Com efeito, considerando-se o valor de K d para o DM-nitrophen de 10 nM e a concentração total de DM-nitrophen e Ca 2 + para ser de 400 mM, a 90% de uma proteína de ligação de cálciocom a K d = 10 uM estará na apoform. Por outro lado, para o estudo de Ca 2 + de ligação para proteínas com a K d &lt;10 uM, é preferível para diminuir a [Ca 2 +]: proporção [DM-nitrophen] de 0,95 para evitar a Ca 2 + a complexação com a apo- de proteína antes da gaiola foto-dissociação. </li><li> Solubiliza-se o composto de referência, K 3 [Fe (III) (CN) 6] ou de Na 2 CrO 4, no mesmo tampão de amostra. </li></ol> 2. Configurando o Experimento <ol><li> A configuração experimental básico é mostrado na Figura 2. </li><li> Usar um orifício para o pino (P 2) para ajustar o diâmetro do feixe de sonda (632 nm saída de laser He-Ne, ~ potência de laser 5 mW) a 1 mm e propagar o feixe sonda através do centro de uma célula colocada em uma temperatura suporte de célula controlada usando um M 1 espelho. </li><li> Use um espelho (M 2) por trás da amostra para centralizar o feixe de provaem um centro de posição detector sensível. </li><li> Focar o feixe de sonda no centro do detector, de tal modo que a diferença de tensão entre as duas melhores diodos e inferior dois diodos, bem como a diferença de tensão entre os dois diodos no lado esquerdo e direito do detector é zero. </li><li> Posteriormente, a forma de um diâmetro do feixe de bomba, uma saída de 355 nm, de Q-switched Nd: YAG, FWHM 5 nseg) usando um orifício de 3 mm (P 1), colocado entre dois espelhos de laser 355 nm. </li><li> Copropagate o feixe de bomba através do centro da tina, como demonstrado na Figura 2. É importante que os dois feixes de laser são propagadas através do centro da célula óptica em forma quase colinear para obter um ângulo de deflexão mensurável e, assim, maior a amplitude de sinal de PBD. Em condições experimentais, o ângulo do cruzamento da sonda e bomba de raios é inferior a 15 °. </li><li> Utilizar um composto de referência para alinhar a sonda e bombafeixe de alcançar um sinal de PBD satisfatório, ou seja, uma relação S / N bom e amplitude PBD estável em longos prazos (~ 100 ms). </li><li> Ajustar a posição da viga de bomba em relação ao feixe de prova por um ajuste incremental de 355 nm espelhos laser. </li><li> Medir a amplitude do sinal de referência de PBD, como a diferença entre dois fotodiodos topo e de fundo sobre o detector sensível à posição. O sinal de PBD devem apresentar um rápido aumento na amplitude de uma escala de tempo rápido (&lt;10 ms) e permanecem estáveis ​​na escala de tempo de 100 mseg, como demonstrado na Figura 3. O tiro disparado à variabilidade da amplitude APO é dentro de 5% da amplitude do sinal e da reprodutibilidade do sinal é afetada principalmente por vibrações. </li><li> Verifique a linearidade da amplitude do sinal de PBD em relação à energia térmica libertada medindo a dependência linear do sinal de PBD na potência do laser de excitação e sobre o número de Einsteins absorvido, e um= (1-10-A), em que A corresponde à absorvância no comprimento de onda de referência de excitação. </li><li> Manter a potência do laser inferior a aproximadamente 1.000 μJ e absorvância da amostra de composto / referência no comprimento de onda de excitação inferior a 0,5 para evitar a absorção de multi e diminuição da potência do feixe de bomba, respectivamente, e garantir a linearidade do sinal de PBD. </li></ol> 3. Medidas PBD <ol><li> Comece com a medição dos vestígios de PBD de referência. Coloque a solução do composto de referência em um 1,0 centímetros por 1,0 cm ou 1,0 cm x 0,5 centímetros de células de quartzo e posicionar a célula no suporte de temperatura controlada. Ambos os comprimentos de trajeto fornecer amplitude comparável PBD. </li><li> Detectar o sinal de referência de PBD, como uma função da temperatura no intervalo de temperatura de 16-35 ° C, com o aumento de temperatura de 3 ° C. </li><li> A cada mudança de temperatura, verifique a posição do feixe de prova no detector sensível posição e reajustar a posição do centro do detector, se necessário. </li><li> Verifique a linearidade do sinal de PBD, como uma função da [(dn / dt) / C p ρ] prazo de acordo com a equação 2. </li><li> Coloque a solução de amostra na mesma célula óptica como para o composto de referência, mantendo a mesma orientação da célula óptica, para a medição de referência. </li><li> Detectar os vestígios de PBD de amostra na mesma gama de temperaturas, como para a referência e verificar a linearidade da amplitude da amostra de PBD em relação ao [(dn / dt) / C p ρ] prazo. </li></ol> 4. Análise de Dados A magnitude da deformação é directamente proporcional à variação de volume devido ao aquecimento da amostra (? V po) e alteração de volume não térmico (? V nonth) de acordo com a Equação 1: <img src="/files/ftp_upload/50969/50969eq1.jpg" alt="Equação 1" /> A amplitude da amostra (A S) E de referência (Ar) PBD sinal podem ser descritas usando as equações 2 e 3, respectivamente. <img src="/files/ftp_upload/50969/50969eq2.jpg" alt="Equação 2" /> <img src="/files/ftp_upload/50969/50969eq3.jpg" alt="Equação 3" /> PBD sinal é directamente proporcional ao parâmetro de resposta do aparelho (K) e o número de Einsteins absorvida (EA). O primeiro termo da equação 2, (dn / dt) (1/ρC p) Q, corresponde à mudança de sinal devido ao calor libertado para o solvente. A dn / dt termo representa a alteração em função da temperatura no índice de refracção, ρ é a densidade do solvente, C p é a capacidade de calor. Todos os parâmetros são conhecidos para a água destilada e podem ser determinadas por uma solução tampão por meio da comparação de um sinal de PBD para o composto de referência em água destilada e em um tampão apropriado. Q é a quantidade de calor Returned para o solvente. O ρ (dn / d ρ) é um termo constante sem unidades que é independente da temperatura no intervalo de temperatura de 10-40 ° C 10. Δn termo abs corresponde à variação do índice de refracção devido à presença de espécies de absorção na solução e é insignificante, se o comprimento de onda do feixe de sonda é deslocada em relação ao espectro de absorção de qualquer espécie em solução. O sinal resultante do composto de referência (Ar) é expresso pela Equação 3 em que E h ν é a energia de fotões de comprimento de onda de excitação, e h ν = 80,5 kcal / mol para 355 nm de excitação. <ol><li> Leve a amplitude do sinal de referência de PBD, como a diferença entre o sinal e pretrigger PBD pós de gatilho, conforme demonstrado na Figura 3. De um modo semelhante, determina a amplitude do jejum (A s rápido) e de fase lenta (A s lento) Do sinal da amostra de PBD. </li><li> Para eliminar o parâmetro de resposta do instrumento, K, escalar a amplitude do sinal da amostra de PBD por a amplitude do sinal de PBD para a referência. A razão entre o sinal da amostra para o sinal de referência dá Equação 4 e pode ser escrita como: <img src="/files/ftp_upload/50969/50969eq4.jpg" alt="Equação 4" /> </li><li> Usando esta equação, determine o calor libertado para a solução (Q) e não térmico mudança de volume (nonth? V) associado a uma reação iniciou-foto da inclinação e interceptação, respectivamente, de um lote de [(A s / A r) E h ν] prazo versus temperatura dependente termo [(dn / dt) (1/ρCp)]. </li><li> Para determinar o volume de reação e variação de entalpia para o rápido eo lento processo, dimensionar o volume observado e variação de entalpia para o rendimento quântico apropriado de acordo com as equações 5-7. <img src = &quot;/ files/ftp_upload/50969/50969eq5.jpg&quot; alt = &quot;Equação 5&quot; /&gt; <img src="/files/ftp_upload/50969/50969eq6.jpg" alt="Equação 6" /> <img src="/files/ftp_upload/50969/50969eq7.jpg" alt="Equação 7" /> Para um processo de vários passos com uma cinética ocorre na escala de tempo entre 10 ms ~ 200 ms, o volume e as alterações de entalpia associada com os passos individuais de reacção pode ser determinada. As amplitudes e os tempos de vida para os passos individuais são analisadas por ajuste dos dados para a função f (t) que descreve o perfil de tempo o volume e as alterações de entalpia. <img alt="Equação 8" src="/files/ftp_upload/50969/50969eq8.jpg" /> onde α 0 corresponde ao A s rápido e α i corresponde a um s lento para cada processo individual e τ i é o tempo de vida das etapas de reação individuais. A partir da dependência da temperatura da constante da taxa de for processos individuais (K i = 1 / τ i) os parâmetros de activação de entalpia e de entropia pode ser facilmente determinada através de curvas de Eyring. </li></ol>

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O princípio físico por trás de métodos fototérmicos é que uma molécula foto-animado dissipa o excesso de energia através de relaxamento vibracional para o estado fundamental, resultando em aquecimento térmico do 1,12 solvente circundante. Para solventes, tais como água, esta produz uma expansão rápida de volume (? V th). Moléculas em estado animado também podem passar por processos fotoquímicos que resultam em mudanças de volume nonthermal (? V nonth) devido à ligação ...

Métodos foto-térmico, como a calorimetria fotoacústica, deflexão do feixe fototérmica (APO), e ralar transitória juntamente com excitação laser de nanossegundos representam uma alternativa poderosa para transitória espectroscopia óptica para estudos resolvido em tempo de curta duração intermediários 1,2. Em contraste com as técnicas ópticas, tais como a absorção transiente e espectroscopia de infravermelho, que acompanhar o perfil do tempo de alterações de absorção do cromóforo circundante; técnicas fototérmicos detectar o tempo-dependência de alterações de calor / volume e, portanto, são ferramentas valiosas para investigar os perfis de tempo de opticamente processos de &quot;Silêncio&quot;. Até agora, calorimetria fotoacústica e ralar transitória tem sido aplicado com sucesso para estudar a dinâmica dos processos de conformação induzida por fotografias, incluindo a migração ligante diatômico em globinas 3,4, ligante com a proteína sensor de oxigênio FixL 5, elétrons e transporte de prótons em heme-oxidases de cobre 6 umaª fotossistema II, bem como foto-isomerização em rodopsina 7 e dinâmica conformacional em cryptochrome 8. Para expandir a aplicação de técnicas de fototérmicos aos sistemas biológicos que faltam um cromóforo e / ou fluoróforo interna, a técnica de PBD foi combinado com a utilização do composto de foto-enjaulado gatilho um aumento na concentração do ligando / substrato dentro de alguns microssegundos ou mais rápido, dependendo no composto enjaulado. Esta abordagem permite o monitoramento da dinâmica e energética de mudanças estruturais associadas com a ligação do ligante / substrato para as proteínas que estão faltando um fluoróforo interna ou cromóforo e em escala de tempo que não são acessíveis por instrumentos stop-flow comerciais. Aqui uma aplicação de PBD para monitorar a termodinâmica do composto gaiola, Ca 2 + DM-nitrophen, foto-clivagem, assim como a cinética de Ca 2 + associação para o domínio C-terminal do sensor de cálcio neuronais de Downfluxo Reguladora Elemento Antagonista modulador (sonho) é apresentado. O Ca 2 + é de Ca-lançado foto 2 + DM-nitrophen dentro de 10 ms e religa a uma gaiola unphotolysed com uma constante de tempo de ~ 300 ms. Por outro lado, na presença de um apoDREAM cinética adicional que ocorre na escala de tempo de milisegundos é observada e reflecte a ligação da proteína ligando. A aplicação do PBD para investigar transições conformacionais em sistemas biológicos tem sido de alguma forma limitada devido às dificuldades instrumentais, por exemplo árdua alinhamento da sonda e feixe de bomba para alcançar um sinal de PBD forte e reprodutível. No entanto, uma concepção meticulosa de uma instrumentação de configuração, um controle preciso da temperatura, e um alinhamento cuidadoso do feixe de sonda e uma bomba de proporcionar um sinal de PBD consistente e robusto que permite a monitorização do volume de resolução por tempo e mudanças de entalpia numa ampla escala de tempo de 10 ms para aproximadamente 200 ms. Além disso, modificções do procedimento experimental para assegurar a detecção de vestígios de amostra e de referência em temperatura idêntica, a composição do tampão, a orientação da célula óptica, a potência do laser, etc reduz significativamente o erro experimental em volumes de reacção medidos e as entalpias.

<table border="0" cellpadding="0" cellspacing="0" width="639">
	<colgroup>
		<col />
		<col />
		<col /> 
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">1-(4,5-Dimethoxy-2-Nitrophenyl)-1,2-Diaminoethane-N,N,N&#39;,N&#39;-Tetraacetic Acid</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">D-6814</td>
			<td style="width:220px;">DM-nitrophen, cage calcium compound, keep stock solutions in dark to prevent photodissociation,</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid)</td>
			<td style="width:84px;">Sigma Adrich</td>
			<td>0909C</td>
			<td>HEPES buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium ferricyanide(III)</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">702587</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:84px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:220px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium chromate</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">307831</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">He-Ne Laser Diode 5mW 635nm </td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">54-179</td>
			<td style="width:220px;">use as a probe beam for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Oscilloscope, </td>
			<td style="width:84px;">LeCroy</td>
			<td style="width:119px;">Wave Surfer 42Xs</td>
			<td style="width:220px;">400 MHz bandwith</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nd:YAG laser</td>
			<td style="width:84px;">Continuum</td>
			<td style="width:119px;">ML II</td>
			<td>pump beam for PBD measurments</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M355; Nd:YAG laser mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">47-324</td>
			<td>laser mirror for 355 nm laser line</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M1 and M2; Laser diode mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">43-532</td>
			<td>visilbe laser flat mirror, wavelength range 300-700 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">P1 and P2; Iris Diaphragm</td>
			<td style="width:84px;">Edmind Optics</td>
			<td style="width:119px;">62-649</td>
			<td style="width:220px;">pin hole to shape the probe and pum beams</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">L1; bi-convex lens</td>
			<td style="width:84px;">Thorlabs</td>
			<td style="width:119px;">LB1844</td>
			<td style="width:220px;">a lens to focus the probe beam at the detector, EFL 50 mm, wavelength range 350 - 2000 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DM, dichroic mirror</td>
			<td style="width:84px;">Thorlab </td>
			<td style="width:119px;">DMLP505</td>
			<td style="width:220px;">a longpass dichroic mirror with a cutoff wavelength of 505 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F1; Edge filter</td>
			<td style="width:84px;">Andower </td>
			<td style="width:119px;">500FH90-25</td>
			<td>a long pass filter with a cutoff wavelength of 500 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Temperature-controlled cuvette holder</td>
			<td style="width:84px;">Quantum Northwest</td>
			<td style="width:119px;">FLASH 300</td>
			<td></td>
		</tr>
	</tbody>
</table>

submillisecond conformational changes in proteins resolved by photothermal beam deflection

Deflexão do feixe Photothermal conjunto com foto-acústica calorimetria e ralar térmica pertence à família de métodos fototérmicos que monitorar o tempo de perfil de volume e entalpia mudanças de luz induzidas mudanças conformacionais em proteínas em microssegundos para milissegundo prazos que não são acessíveis através de parada tradicional -fluxo de instrumentos. Além disso, uma vez que as alterações globais de volume e / ou entalpia são sondados, estas técnicas podem ser aplicadas às proteínas e outras biomacromoléculas que carecem de um fluoróforo e um rótulo ou cromóforo. Para monitorar a dinâmica e energética de mudanças estruturais associadas ao Ca 2 + ligação a transdutores de cálcio, tais sensores de cálcio neuronais, um composto de cálcio enjaulado, DM-nitrophen, é empregado para foto-trigger um aumento rápido (τ &lt;20 ms) em cálcio livre concentração eo volume associado e entalpia mudanças são detectados usando feixe fototérmica técnica deflexão.

Um exemplo representativo de PBD traça de Ca 2 + a foto-libertação de Ca2 + DM-nitrophen é mostrado na Figura 3. A fase rápida corresponde à foto-clivagem de Ca 2 + DM-nitrophen e Ca 2 + de libertação, enquanto que a fase lenta reflecte Ca 2 + ligação para a gaiola nonphotolysed. O gráfico da amplitude de PBD amostra para a fase rápida e lenta escalado para a amplitude do composto de referência, em função do factor de dependia da temp...

Watch this Scientific Journal Video about Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection at JoVE.com

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection | Chemistry | Video

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Aqui descrevemos uma aplicação da técnica de deflexão do feixe fototérmica em combinação com um composto de cálcio enjaulado, DM-nitrophen, para monitorar microssegundo e milissegundo dinâmica e energética de mudanças estruturais associadas com a associação de cálcio para um sensor de cálcio neuronal, Downstream Elemento Regulador Antagonista modulador .

Submillisecond alterações na conformação das proteínas resolvidas por Photothermal Boca deflexão

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

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8.9K visualizações.  Florida International University.  Aqui descrevemos uma aplicação da técnica de deflexão do feixe fototérmica em combinação com um composto de cálcio enjaulado, DM-nitrophen, para monitorar microssegundo e milissegundo dinâmica e energética de mudanças estruturais associadas com a associação de cálcio para um sensor de cálcio neuronal, Downstream Elemento Regulador Antagonista modulador . 

Vídeo: Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Determinando os planos de ligação de gelo de proteínas anticongelantes pela afinidade do plano de gelo baseado em fluorescência

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Química

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

O uso de cromatografia gasosa para analisar composicional Alterações de ácidos graxos no tecido de fígado de rato durante a gravidez

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Radiomarcação e quantificação dos níveis celulares de Fosfoinositídeos por Cromatografia Líquida de Alta Eficiência acoplado Fluxo de Cintilação

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

In Situ Caracterização de Proteínas hidratadas em água por SALVI e TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

Um ensaio de HS-MRM para a quantificação de proteínas de célula hospedeira em biofármacos de proteínas por espectrometria de massa líquida cromatografia iônica mobilidade QTOF

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

Tempo-resolvido compostos orgânicos de Photophysical caracterização dos Triplet-colheita em um livre de oxigênio ambiente usando uma câmera de iCCD

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

DNA-magnético partícula análise por dispersão de luz dinâmica e eletroforético de vinculação

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Co de expressão de várias proteínas da fusão fluorescente quimérico de forma eficiente em plantas

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&lt;sup&gt;&amp;#8722;&lt;/sup&gt;

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

Imagem de fotoelétron de ânions ilustrado por 310 Nm destacamento de F−

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

Melhorando a extrusão de alta viscosidade de microcristais de tempo-resolvido Serial Femtosecond cristalografia em Lasers de raio-x

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...