Este trabalho foi financiado por doações e AG027818 AG030709 de NIH / NIA, 2005/2E da L. Larry Hillblom Foundation, IIRG-07-58334 da Associação Alzheimer, e 07-65798 da Califórnia Departamento de Saúde Pública.

1. Preparação de peptídeos <ol><li> Pesar ~ 100-200 mg de peptídeo liofilizado usando uma microbalança e transferência para rotulados, revestido a silicone, tubos de microcentrífuga de baixa adsorvente. Aqui, nós usamos seqüências de humanos de Aβ40, Aβ42, calcitonina e GRF. </li><li> Aqui, usamos peptídeos pré-tratados com 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) para obter homogênea, agregar-free preparativos. Esta etapa é necessária porque pré-formados agregados induzir a agregação rápida de proteínas amiloidogênicas, que resultam em reprodutibilidade pobres entre os experimentos 5. Outros métodos, como a filtração ea SEC também pode ser usado para obter agregados sem preparativos para picup 6. </li><li> Para tratar os peptídeos com HFIP, pré-resfriar o recipiente com gelo dentro HFIP um exaustor usando proteção adequada (HFIP é volátil e tóxico). Resfriamento de uma garrafa de 250 ml normalmente exige 10-15 min. Adicionar HFIP de pré-refrigerados contendo tubos lyophilizates peptídeo para obter uma concentração de peptídeo nominal de 0,5 mM. </li><li> Sonicate as soluções em um peptídeo sonicador banho-maria por 5 minutos em temperatura ambiente. </li><li> Vortex suavemente e incubar os tubos durante 30 min à temperatura ambiente. </li><li> Relaxar os tubos no gelo (para 1 min), e dividir as soluções em 10-50-mL em alíquotas de 0,6 ml rotulados de baixa adsorvente tubos. </li><li> Remover HFIP por evaporação sob uma ligeira corrente de nitrogênio, ou deixar os tubos abertos na capela durante a noite. Para a evaporação durante a noite, coloque os tubos abertos em um rack e cobri-los com uma grande folha de Kimwipes para evitar poeira e contaminação por partículas. </li><li> Enxugar o HFIP restantes no vácuo em um liofilizador, ou um concentrador de centrífuga por 30 min, ou em um exsiccator ligado a uma entrada de vácuo por 2 h. O produto final será um filme peptídeo na parte inferior dos tubos de microcentrífuga. Se devidamente ressecada, os tubos podem ser armazenados por longos períodos hermético (meses) a -20 ou -80 ° C. </li></ol> 2. Solubilizar os peptídeos HFIP-tratados e foto cross-linking <ol><li> Antes de solubilizar os peptídeos HFIP-tratado para cross-linking reações, é preciso preparar o cross-linking e reagentes de extinção. </li><li> Pesar persulfato de amônio (APS, o Sr. 228,2 g / mol) e preparar uma solução de 20 mm em 10 mM fosfato de sódio, pH 7.4. Misture usando um vortex até que a solução é clara. </li><li> Prepare uma solução mM de Tris (2,2-bipyridyl) dichlororuthenium hexa (II) (RuBpy, deputado 748,63 g / mol) em 10 mM fosfato de sódio, pH 7.4. Misture usando um vortex e verificar a completa dissolução. Proteger o tubo de luz usando uma folha de alumínio. </li><li> Para SDS-PAGE após análise cross-linking, um reagente quenching conveniente é de 5% β-mercaptoetanol em 2 × SDS-PAGE tampão de amostra. Alternativamente, um M ditiotreitol (DTT, o Sr. 154,5 g / mol) em água deionizada ou um tampão adequado pode ser usado. </li><li> Filmes HFIP tratados com peptídeo são dissolvidos em NaOH diluída primeiro e depois tampão fosfato de sódio é adicionado para obter concentração de peptídeo ~ 30 mM. Adicionar 60 mM NaOH seguido de água deionizada no tubo contendo o filme de tal forma que peptídeo NaOH e água constituem 10 e 45% do volume final, respectivamente. Raspe o filme peptídeo fora das paredes dentro do tubo de microcentrífuga, utilizando a ponta, misture pipetando cima e para baixo, e sonicate por 5 minutos em um sonicador banho-maria. </li><li> Adicionar 45% de fosfato de sódio 20 mM, pH 7,4, mix de pipetagem, e centrifugar a 16.000 g por 10 min. Separe alíquotas de descruzar-linked peptídeos misturado com o reagente de têmpera para controles negativos. </li><li> Ajustar a câmera do obturador demora a 1 s. Às vezes maior irradiação, extensas reações radicais podem causar degradação de proteínas. Carregar o obturador da câmera. Tempo de irradiação pode precisar de ser otimizado quando se utiliza o método com uma nova proteína. </li><li> Uma reação típica é picup realizada em um volume de reação 20 l. Transferência de 18 mL da solução de peptídeo em uma parede fina, clara, tubo de PCR de 0,2 ml. </li><li> Para a solução de peptídeo, adicione 1 mL RuBpy seguido por uma APS mL e misturar os reagentes por pipetagem (a concentração final de RuBpy APS e na mistura de reação será 0,05 e 1 mM, respectivamente). </li><li> Colocar rapidamente o tubo de reação dentro de um frasco de vidro de 1,8 mL. Coloque o frasco dentro do fole anexado na frente do corpo da câmera. Anexar o protetor de lente e pressione o botão do obturador para que a amostra é irradiada por 1 s no interior do fole. </li><li> Após a irradiação da amostra, assumir rapidamente o frasco fora do fole e do tubo de PCR fora do frasco. Rapidamente saciar a reação pela adição de um TDT mL ou 10 mL tampão de amostra reduzindo PAGE. Repita a reação para cada alíquota peptídeo. </li><li> As misturas de reação pode agora ser congeladas a -20 ° C para o armazenamento por um período de 7 dias ou mantidos em gelo antes da análise por SDS-PAGE e coloração de prata. </li></ol> 3. SDS-PAGE e tira-manchasde produtos cross-linked peptídeo <ol><li> Rotina SDS-PAGE e coloração prata-são realizadas para visualizar cruzada peptídeos. </li><li> Carga de 5 mL da mistura de reação por pista para obter ~ 130 pmol de peptídeo por pista. </li><li> Incluem quantidades similares de descruzar-linked peptídeos para comparação. Também incluem uma escada de proteína padrão de aproximação visual de bandas de peso molecular de peptídeos. </li><li> Executar o gel utilizando um aparelho de eletroforese em gel padrão. Nós usamos o sistema de XCell SureLock Mini-Cell da Invitrogen e realizar a coloração de prata de acordo com a publicação Invitrogen IM-1002, Novex Pre-Cast Guia de eletroforese em gel. </li></ol> Parte 4: Resultados Representante (Figura 1) <img src="/files/ftp_upload/old/1071_2009_1_19_13/Result no demarcation.png" alt="Figura 1" /> Figura 1: géis de poliacrilamida mostrando uncross-linked (-) e foto-cross-linked (+) GRF, calcitonina, Aβ40 e Aβ42 SDS-PAGE e coloração prata-análises de picup gerado Aβ40 oligômeros mostram intensidades banda aproximadamente igual de monômero através tetrâmero, seguido por uma acentuada diminuição na abundância de oligômeros maior 7. Descruze-linked Aβ40 migra com um deputado consistente com a de monômero 7. Aβ42 mostra uma distribuição de tamanho distintas oligômero. Aβ42 compreendem oligômeros 2-3 grupos de 8. O primeiro grupo, através de monômero trímero, exibe a diminuir de intensidade com a ordem crescente de oligômero. No segundo grupo, a distribuição de Gauss-como é observada entre tetrâmero e heptamer, com um máximo em pentâmero e hexâmero. O terceiro grupo, que não é mostrado aqui, contém oligômeros do Sr. ~ 30,000-60,000 Da 8. Estes oligômeros maior Mr normalmente são observados usando SEC isolada LMW Aβ42 mas não peptídeo preparada por filtração ou tratamento HFIP 6. Descruze-linked Aβ42 produz predominantemente duas bandas, uma banda de monômero e uma banda trímero / tetrâmero amplo, que é um artefato induzida por SDS 5,9. Calcitonina produz uma distribuição de tamanho de oligômero que diverge fortemente de uma diminuição monótono exponencial do monômero região através tetrâmero, sugerindo pré-existência de dímeros, trímeros, tetrâmeros e 7. O controle de polipeptídeo, GRF, produz uma distribuição de oligômero monotônica desde dímeros através de hexâmero de tal forma que os montantes aparente relativa de oligômeros diminuir exponencialmente com o aumento da massa molecular. Em outros experimentos, oligômeros mais altas também poderiam ser visualizadas 7. O número exato e intensidades relativas das bandas de oligômero pode variar um pouco entre os experimentos, dependendo da concentração de proteína real, a quantidade de proteína carregado no gel, eo tempo utilizado para a etapa de desenvolvimento no protocolo de coloração prateada.

<ol>
	<li>Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+study+of+metastable+amyloidogenic+protein+oligomers+by+photo-induced+cross-linking+of+unmodified+proteins.">Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.</a> Methods Enzymol. 413, 217-236 (2006).</li><li>Fancy, D. A., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+for+the+analysis+of+protein-protein+interactions:+rapid+and+efficient+cross-linking+triggered+by+long+wavelength+light.">Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.</a> Proc. Natl. Acad. Sci. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</li><li>Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+(A&#946;)+assembly:+A&#946;40+and+A&#946;42+oligomerize+through+distinct+pathways.">Amyloid &#946;-protein (A&#946;) assembly: A&#946;40 and A&#946;42 oligomerize through distinct pathways.</a> Proc. Natl. Acad. Sci. USA. 100, 330-335 (2003).</li><li>Hepler, R. W., Grimm, K. M., Nahas, D. D., Breese, R., Dodson, E. C., Acton, P., Keller, P. M., Yeager, M., Wang, H., Shughrue, P., Kinney, G., Joyce, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+state+characterization+of+amyloid+&#946;-derived+diffusible+ligands.">Solution state characterization of amyloid &#946;-derived diffusible ligands.</a> Biochemistry (Mosc). 45, 15157-15167 (2006).</li><li>Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+photochemical+cross-linking&#8212;a+new+tool+for+studies+of+metastable,+amyloidogenic+protein+assemblies).">Rapid photochemical cross-linking&#8212;a new tool for studies of metastable, amyloidogenic protein assemblies).</a> Acc. Chem. 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Picup foi desenvolvido originalmente para estudar complexos de proteína estável 2. O método foi aplicado mais tarde para estudo quantitativo de assembléias amilóide metaestável proteína, incluindo Aâ 10, prion e doenças associadas ao PrP Sc 11, e α-sinucleína 12. Os fatores mais importantes que devem ser considerados ao projetar um experimento picup estão a estequiometria reagente, tempo de irradiação, eo procedimento de preparação da amostra. As duas primeiras questões podem exig...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dolan-Jenner 200-W incandescent lamp</td>
<td>Other</td>
<td>Dolan-Jenner Industries</td>
<td>Model 170-D</td>
<td>The heat generated by the lamp does not affect samples for short incubation periods.</td>
</tr>
<tr>
<td>35-mm SLR camera body</td>
<td>Tool</td>
<td>Pentax</td>
<td>SP500 model</td>
<td>In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.</td>
</tr>
<tr>
<td>Clear, thin walled PCR tubes</td>
<td>Other</td>
<td>Eppendorf</td>
<td>951010006 supplied by Fisher L22-003-24</td>
<td>&nbsp;</td>
<tr>
<td>Glass vials (1.8 mL)</td>
<td>Other</td>
<td>Kimble Chromatography</td>
<td>60940A 2, supplied by Fisher 03-340-60</td>
<td>&nbsp;</td>
<tr>
<td>GRF</td>
<td>Reagent</td>
<td>Bachem</td>
<td>H-3695</td>
<td>&nbsp;</td>
<tr>
<td>HFIP</td>
<td>Reagent</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>A&beta;40 and A&beta;42</td>
<td>Reagent</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calcitonin</td>
<td>Reagent</td>
<td>American Peptide</td>
<td>22-1-10</td>
<td>&nbsp;</td>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. </td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7154-25 ML</td>
<td>Can be used when SDS-PAGE analysis is performed.</td>
</tr>
<tr>
<td>Novex Tricine SDS Sample Buffer (2x)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10-20%)</td>
<td>Other</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10x )</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Silver Express Staining Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6100</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides

O conjunto de proteínas amiloidogênicas em oligômeros tóxicos é um evento seminal na patogênese de doenças proteína misfolding, incluindo Alzheimer, Parkinson e doenças de Huntington, esclerose amiotrófica hereditária lateral, e diabetes tipo 2. Devido à natureza metaestável destes conjuntos de proteínas, é difícil avaliar a sua distribuição de tamanho de oligômero quantitativamente usando métodos clássicos, tais como eletroforese, cromatografia de espalhamento de luz, fluorescência, ou dinâmico. Oligômeros de proteínas amiloidogênicas existir como misturas metaestável, em que os oligômeros se dissociam em monômeros e associar em conjuntos maiores simultaneamente. Picup estabiliza populações oligômero por covalente cross-linking e quando combinado com métodos de fracionamento, como eletroforese em gel de sulfato de sódio dodecil de poliacrilamida (SDS-PAGE) ou tamanho-exclusão cromatografia (SEC), picup fornece instantâneos das distribuições de tamanho de oligômero que existia antes de cruz -linking. Assim, picup permite a visualização e análise quantitativa das populações de proteína metaestável e pode ser usado para monitorar a montagem e decifrar as relações entre modificações seqüência e oligomerização<sup&gt; 1</sup&gt;. Mecanicamente, picup envolve foto-oxidação de Ru<sup&gt; 2 +</sup&gt; Num (bipyridyl) Ru tris complexo (II) (RuBpy) a Ru<sup&gt; 3 +</sup&gt; Por irradiação com luz visível na presença de um receptor de elétrons. Ru<sup&gt; 3 +</sup&gt; É um oxidante de um elétron forte, capaz de abstrair um elétron de uma molécula de proteína vizinhos, gerando uma proteína radical<sup&gt; 1,2</sup&gt;. Os radicais são instáveis ​​e altamente reativos e, portanto, as espécies desaparecem rapidamente através de uma variedade de reações intra e intermoleculares. Um radical pode utilizar a energia de alta de um elétron desemparelhado reagir com outro monômero formando uma proteína dimérica radical, que, posteriormente, perde um átomo de hidrogênio e forma uma estável, dímero covalentemente ligadas. O dimer pode então reagir ainda mais através de um mecanismo semelhante com monômeros ou dímeros para formar oligômeros de ordem superior. Vantagens de parente picup para outros foto-química ou cross-linking métodos<sup&gt; 3,4</sup&gt; Incluem a exposição (≤ 1 s) curta para não-destrutiva da luz visível, sem necessidade de<i&gt; Pré facto</i&gt; Modificação da seqüência nativa, e de comprimento zero covalente cross-linking. Além disso, permite picup ligação cruzada de proteínas dentro pH de largura e faixas de temperatura, incluindo parâmetros fisiológicos. Aqui, demonstramos a aplicação de picup cross-linking de três proteínas amiloidogênicas a 40 - e 42 resíduos de amilóide β proteínas variantes (Aβ40 e Aβ42) e calcitonina, e uma proteína de controle, fator de crescimento hormônio liberador (GRF).

Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Foto-induzida ligação cruzada de proteínas não modificadas (picup) permite a caracterização da distribuição de tamanho de oligômero em misturas de proteínas metaestável. Demonstramos aplicação de picup a três peptídeos representante amiloidogênicas a 40 - e 42 resíduos de formas de amilóide β-proteína, e calcitonina, peptídeo e um controle do fator de crescimento hormônio liberador.

Foto-Induzida Cross-Linking de Proteínas Unmodified (picup) Aplicada à Peptídeos amiloidogênicas

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

photo-induced-cross-linking-unmodified-proteins-picup-applied-to

Research

JoVE Journal

Biology

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

21.2K visualizações.  University of California, Los Angeles. Foto-induzida ligação cruzada de proteínas não modificadas (picup) permite a caracterização da distribuição de tamanho de oligômero em misturas de proteínas metaestável. Demonstramos aplicação de picup a três peptídeos representante amiloidogênicas a 40 - e 42 resíduos de formas de amilóide β-proteína, e calcitonina, peptídeo e um controle do fator de crescimento hormônio liberador.

Vídeo: Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Separação eletroforética de proteínas

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Biologia

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Coloração Proteínas em Gel

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Um método HPLC Lectin enriquecer Peptídeos seletivamente glicosilada de Complexo Amostras Biológicas

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolamento de proteínas lábeis Multi-Complexos por In vivo Controlled Cellular Cross-Linking and Imuno-magnética cromatografia de afinidade

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Microdissecção a laser Aplicada ao perfil de expressão gênica de subconjunto de células a partir do Drosophila Asa Disco

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening expressão quantitativa e Purificação Aplicada à recombinante Dissulfeto ricos em proteínas veneno produzido em<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

A purificação cromatográfica do não-polipéptidos recombinantes Elastina semelhantes e suas fusões com Peptídeos e Proteínas de<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

Uso de MALDI-TOF Espectrometria de Massa e um banco de dados personalizado para caracterizar bactérias indígenas para uma caverna ambiente único (Kartchner Caverns, AZ, EUA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Um método para selecionar Aptamers de comutação Estrutura Aplicado a um colorimétrico nanopartículas de ouro Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Enriquecimento de Proteínas da Matriz Extracelular a partir de tecidos e digestão em péptidos para Análise de Espectrometria de Massa

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...