This work was supported by the Lehigh University Start-up Package. We thank Dr. Walter Fast for providing the GST-PAD4 plasmid for protein expression.

1. PAD4 Transformação, expressão e purificação <ol><li> Para PAD4 Transformação, transformar o plasmídeo pGEX contendo PAD4 GST fusão em E. quimicamente competente Coli (células BL21 (DE3)), as células para a expressão de proteínas utilizando o seguinte procedimento. Prepare (cloreto de cálcio) quimicamente competentes E. Coli (células BL21 (DE3)), as células de acordo com protocolos padrão. <ol><li> Descongelar 50 ul de previamente preparada quimicamente competentes BL21 (DE3) células em gelo e misturar com 1 ul do plasmídeo pGEX contendo o gene PAD4 num tubo de cultura de 5 mL. Incubar a mistura em gelo durante 10 minutos, enquanto agitando a cada 2 min. </li><li> Calor chocar as células colocando a mistura em um banho de água a 42 C ° durante 40 seg. Colocar imediatamente a mistura de células-plasmídeo para trás em gelo durante 2 min para permitir que as células para recuperar. Adicionar 1 ml de caldo LB estéril à mistura e colocar em gelo durante 1 min. </li><li> Incubar o calor células chocados a 37 ° C, agitação a 250 rpm durante 1hr. Pipetar 75 ul das células transformadas numa placa de ágar resistente à ampicilina e incubar a 37 ° C durante 15 h. Placa armazenar a 4 ° C. </li></ol></li><li> PAD4 Expressão. <ol><li> Picareta 1 colónia de células BL21 (DE3) a partir da placa de ágar e ampicilina-se em 5 ml de LB contendo ampicilina 1x. Colocar na incubadora e agite O / N a 37 ° C. </li><li> Transferir os 5 ml de LB (kits) em 1 L de LB estéril contendo 1x tri-hidrato de ampicilina (PM 403,45 g / mol). Ponto de crescimento num incubador com agitação a 37 ° C. Monitorar a OD 600 do crescimento. Quando o crescimento atinge uma DO600 de 0,3, mova o crescimento em 16 ° C agitando incubadora. </li><li> Ao atingir uma DO600 de 0,6, induzir as células com 0,3 mM isopropylthiagalactoside (IPTG, MW 238,30 g / mol). Permitir que as células agitar por 15 horas a 16 ° C. </li><li> Células colheita por centrifugação a 4000 xg durante 20 min a 0 ° C. Decantar o sobrenadante e pellet loja a -80 ° C. </li></ol></li><li> PAD4 Purificação <ol><li> Re-suspender o sedimento contendo o PAD4 expressa em BL21 (DE3) células num tampão de NaCl 50 mM (PM 58,44 g / mol), 300 mM de NaH 2 PO 4 (PM 119,98 g / mol), 10 mM de imidazole (MW 68,077 g / mol), 0,1 mM de fluoreto de fenilmetilsulfonilo (PMSF, MW 174,94 g / mol) e 1 mM de ditiotreitol (DTT, 154,25 g / mol), pH = 8,0. </li><li> Lisar as células por sonicação durante 15 min a 4 ° C. Após sonicação, centrifugar o lisado celular a 20000 xg durante 20 min a 0 ° C. Deitar fora e salvar sobrenadante. Lote do sobrenadante com glutationa (GSH) esferas de agarose durante 30 minutos à TA. </li><li> Drenar o sobrenadante das esferas de GST / coluna por acção da gravidade. Lavar as pérolas com 4 x 25 ml de 1x PBS (solução salina tamponada com fosfato, pH = 8,0) à temperatura ambiente. </li><li> Eluir PAD4 com 2 x 10 ml de tampão de eluição, 50 mM de tris (hidroximetil) aminometano (Tris Base, MW 121,14 g / mol), 10 mM de glutationa (GSH, MW 307,32 g / mol), pH = 8,0. </li><li> Concentre PAD4 usando 100k MW corte-off tubos de centrífuga e centrifugar a 4000 xg durante 20 min a 4 ° C. Aliquota de proteína em 200 ul volumes de 1,0 ml em tubos de microcentrífuga e armazenar a -80 ° C. </li></ol></li></ol> 2. Preparar soluções estoque para amortecedores <ol><li> Pesam-se cloreto de sódio (NaCl, PM 58,44 g / mol) e preparar uma solução 2 M. Misturar a solução até que clara. </li><li> Pesar Tris (hidroximetil) aminometano (Tris Base, MW 121,14 g / mol) e preparar uma solução 2 M, pH = 8,0. Misturar a solução até que clara. </li><li> Pesam-se di-hidrato de cloreto de cálcio (CaCl 2 2H 2 O, MW 147,01 g / mol) e preparar uma solução 500 mM. Misturar a solução até que clara. </li><li> Pesar de Tris (2-carboxietil) fosfina (TCEP, MW 250,19 g / mol) e preparar uma solução 200 mM. Misturar a solução até clara e armazenar a -20 ° C. </li><li> Pesar ditiotreitol (DTT, MW 154,25 g / mol) e preparar uma solução 1 M. Misturar a solução até clara e armazenar a -20 ° C. </li><li> Prepararuma solução de 0,5% de Triton X-100. </li><li> Pesar o ácido etilenodiaminotetracético (EDTA, MW 292,24 g / mol) e preparar uma solução 100 mM. Misturar a solução até que clara. </li><li> Pesar Z- Arg-Arg-7- amido- metilcumarina cloridrato de 4- (ZRcoum, MW 621,69 g / mol) e preparar uma solução a 10 mM em sulfóxido de dimetilo (DMSO). </li></ol> 3. PAD4 Ensaio a 37 ° C <ol><li> De 10 ações ZRcoum mM, preparar uma solução de 125 mM de ZRcoum na água. Esta solução 125 uM ZRcoum será Solução A. </li><li> Preparar um tampão de NaCl a 62,5 mM, Tris 62,5 mM, 12,5 mM de CaCl2, 6,25 mM de DTT, e PAD4 5 uM (pH = 8,0). Este será Solução B. </li><li> Preparar um tampão de NaCl a 62,5 mM, Tris 62,5 mM, 12,5 mM de CaCl2, e 6,25 mM de DTT (pH = 8,0). Este será Solução C. </li><li> Pesar tripsina, cristalino (a partir de pâncreas bovino) e preparar um 10 mg / ml em 100 mM de EDTA. Misturar a solução até clara e armazenar a -20° C. Este será Solução D. </li><li> Obter uma placa de 96 poços e designar um número de poços para controlos (sem PAD4) e os poços de ensaio (+ PAD4). </li><li> Pipeta 160 mL de solução B para poços de teste e pipeta de 160 mL de solução de C em poços de controlo. Incubar durante 15 min a 37 ° C. </li><li> Pipetar 40 ul de solução A em todos os poços e incubar a 37 ° C durante 45 min. </li><li> Pipetar 10 ul de dH2O a metade dos poços de controlo e metade dos poços de teste. Para a outra metade dos poços de controlo e controlos de ensaio e pipetas 10 mL de solução de D. Incubar durante 10 min a 37 ° C. </li><li> Obter uma leitura de fluorescência em um leitor multimodal com um comprimento de onda de excitação de 345 nm e um comprimento de onda de emissão de 465 nm. </li></ol> 4. Ensaio PAD4 à TA <ol><li> De 10 ações ZRcoum mM, preparar uma solução de 50 mM de ZRcoum na água. Esta solução ZRcoum 50 uM será Solução A. </li><li> Preparar um tampão de Na 100 mMCl, 100 mM Tris, 20 mM de CaCl2, 2 mM de TCEP, 0,02% de Triton X-100, e 8 PAD4 uM (pH = 8,0). Este será Solução B. </li><li> Preparar um tampão de 100 mM de NaCl, 100 mM Tris, 20 mM de CaCl2, 2 mM de TCEP, 0,02% e Triton X-100 (pH = 8,0). Este será Solução C. </li><li> Pesar tripsina, cristalino (a partir de pâncreas bovino) e preparar um 10 mg / ml em 100 mM de EDTA. Misturar a solução até clara e armazenar a -20 ° C. Este será Solução D. </li><li> Obter uma placa de 384 poços e poços para designar controles (sem PAD4) e poços de teste (+ PAD4). </li><li> Pipetar 25 mL de solução B para poços de teste e pipeta de 25 mL de solução de C em poços de controlo. Permitir tampão com / sem PAD4 a incubar nos poços durante 20 min à TA. </li><li> Pipetar 25 ul de solução C em todas as cavidades e incubar durante 45 min à temperatura ambiente. </li><li> Pipetar 10 ul de dH2O a metade dos poços de controlo e metade dos poços de teste. Para a outra metade dos poços de controlo e controlos de ensaio, pipetae 10 ul de solução de D. Incubar à temperatura ambiente durante 10 min. </li><li> Obter uma leitura de fluorescência em um leitor multimodal com um comprimento de onda de excitação de 345 nm e um comprimento de onda de emissão de 465 nm. </li></ol> 5. PAD4 tempo do curso ensaios a 37 ° C e RT <ol><li> De 10 ações ZRcoum mM, preparar uma solução de 125 mM de ZRcoum na água. Esta solução 125 uM ZRcoum será Solução A. </li><li> Preparar um tampão de NaCl a 62,5 mM, Tris 62,5 mM, 12,5 mM de CaCl2, 6,25 mM de DTT, e PAD4 5 uM (pH = 8,0). Este será Solução B. </li><li> Pesar tripsina, cristalino (a partir de pâncreas bovino) e preparar um 10 mg / ml em 100 mM de EDTA. Misturar a solução até clara e armazenar a -20 ° C. Este será Solução C. </li><li> Obter duas placas de 96 poços e pipeta de 160 mL de solução B em # quantidade de poços (número de poços vai depender de quantos pontos de tempo). </li><li> Incubar uma placa de 96 poços, a 37 ° C. Incubar a outra placa de 96 cavidades, à TA. </li><li> Pipette 40 ul de solução A em todos os poços em ambas as placas de 96 poços e imediatamente pipeta 10 ul de solução C em t = 0 o mínimo de poços. Continue a adição de 10 ul de solução C em pontos de tempo específicos até que todos os poços foram temperada. </li><li> Obter uma leitura de fluorescência em um leitor multimodal com um comprimento de onda de excitação de 345 nm e um comprimento de onda de emissão de 465 nm. </li></ol> 6. Ensaio de Inibição PAD4 à TA <ol><li> De 10 ações ZRcoum mM, preparar uma solução 83 mM de ZRcoum na água. Esta solução ZRcoum 83 uM será Solução A. </li><li> Pesar Cl-amidina (MW 424,8 g / mol) e preparar uma solução stock de 100 uM. Este será Solução B. </li><li> Preparar um tampão de 100 mM de NaCl, 100 mM Tris, 20 mM de CaCl2, 2 mM de TCEP, 0,02% de Triton X-100, e 8 uM PAD4 (pH = 8,0). Este será Solução C. </li><li> Preparar um tampão de 100 mM de NaCl, 100 mM Tris, 20 mM de CaCl2, 2 mM de TCEP, 0,02% e Triton X-100 (pH = 8,0). Thé será Solution D. </li><li> Pesar tripsina, cristalino (a partir de pâncreas bovino) e preparar um 10 mg / ml em 100 mM de EDTA. Misturar a solução até clara e armazenar a -20 ° C. Este será Solução E. </li><li> Obter uma placa de 384 poços e poços para designar controles (sem PAD4), poços de teste (+ PAD4) e poços de inibição (PAD4 + Cl-amidina). </li><li> Pipetar 25 mL de solução C em poços de teste e pipeta de 25 mL de solução de D em poços de controlo. Permitir tampão com / sem PAD4 a incubar nos poços durante 20 min à TA. </li><li> Pipetar 10 ul de solução B em poços de inibição e incubar durante 20 min a RTe. </li><li> Pipetar 15 ul de solução A em todos os poços e incubar durante 45 min à temperatura ambiente. </li><li> Pipetar 10 mL de solução E para todos os poços. Incubar à temperatura ambiente durante 10 min. </li><li> Obter uma leitura de fluorescência em um leitor multimodal com um comprimento de onda de excitação de 345 nm e um comprimento de onda de emissão de 465 nm. </li></ol> 7. Ensaio Lisado celular brutoà TA <ol><li> Após a colheita de células PAD4 crescimento de 1 L, ressuspender as células em 10 ml de 50 mM Tris, 50 mM de NaCl, 0,1 mM de fluoreto de fenilmetilsulfonilo (PMSF, MW 174,94 g / mol) e 1 mM de ditiotreitol (DTT, 154,25 g / mol), pH = 8,0. </li><li> Lisar as células por sonicação durante 15 min a 4 ° C. Centrifuga-se o lisado celular a 20000 xg durante 20 min a 0 ° C. Concentra-se o sobrenadante claro até metade do volume por centrifugação a 4.000 rpm a 4 ° C utilizando 100k MW tubos de centrífuga de corte. </li><li> De 10 ações ZRcoum mM, preparar uma solução de 250 mM de ZRcoum na água. Esta solução 250 uM ZRcoum será Solução A. </li><li> Preparar um tampão de 100 mM de NaCl, 100 mM Tris, 20 mM de CaCl2, e TCEP 2 mM (pH = 8,0). Este será Solução B. </li><li> Pesar tripsina, cristalino (a partir de pâncreas bovino) e preparar um 10 mg / ml em 100 mM de EDTA. Misturar a solução até clara e armazenar a -20 ° C. Este será Solução C. </li><li> Obter um preto placa de 384 poços e designcomeu poços para controles (não lisado celular) e poços de teste (+ lisado celular). </li><li> Pipetar 100 mL de solução B para todos os poços e pipeta 60 mL de lisado de células nos poços de teste. Incubar durante 20 min à TA. </li><li> Pipetar 40 ul de solução A em todos os poços e incubar durante 45 min à temperatura ambiente. </li><li> Pipetar 10 ul de solução C a todos os poços. Incubar à temperatura ambiente durante 10 min. </li><li> Obter uma fluorescência leitura em leitor multimodal com um comprimento de onda de excitação de 345 nm e um comprimento de onda de emissão de 465 nm. </li></ol>

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Devel. 12, 616-627 (2009).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+p53+target+gene+expression+by+peptidylarginine+deiminase+4.">Regulation of p53 target gene expression by peptidylarginine deiminase 4.</a> Mol. Cell Biol. 28, 4745-4758 (2008).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticancer+peptidylarginine+deiminase+(PAD)+inhibitors+regulate+the+autophagy+flux+and+the+mammalian+target+of+rapamycin+complex+1+activity.">Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity.</a> J. Biol. Chem. 287, 25941-25953 (2012).</li><li>Slack, J. L., Causey, C. P., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+deiminase+4:+a+target+for+an+epigenetic+cancer+therapy.">Protein arginine deiminase 4: a target for an epigenetic cancer therapy.</a> Cell. Mol. Life Sci. 68, 709-720 (2011).</li><li>Luo, Y., Knuckley, B., Lee, Y. H., Stallcup, M. R., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluoroacetamidine-based+inactivator+of+protein+arginine+deiminase+4:+design,+synthesis,+and+in+vitro+and+in+vivo+evaluation.">A fluoroacetamidine-based inactivator of protein arginine deiminase 4: design, synthesis, and in vitro and in vivo evaluation.</a> J. Am. Chem. Soc. 128, 1092-1093 (2006).</li><li>Knuckley, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+specificity+and+kinetic+studies+of+PADs.">Substrate specificity and kinetic studies of PADs.</a> Biochemistry. 1, 4852-4863 (2010).</li><li>Knipp, M., Vasak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+96-well+microtiter+plate+assay+for+the+determination+of+enzymatically+formed+citrulline.">A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline.</a> Anal. Biochem. 286, 257-264 (2000).</li><li>Knuckley, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluopol-ABPP+HTS+assay+to+identify+PAD+inhibitors.">A fluopol-ABPP HTS assay to identify PAD inhibitors.</a> Chem. Commun. 46, 7175-7177 (2010).</li><li>Bicker, K. L., Subramanian, V., Chumanevich, A. A., Hofseth, L. J., Thompson, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+citrulline:+development+of+a+phenylglyoxal-based+probe+to+visualize+protein+citrullination.">Seeing citrulline: development of a phenylglyoxal-based probe to visualize protein citrullination.</a> J. Am. Chem. Soc. 134, 17015-17018 (2012).</li><li>Wang, Q., Priestman, M. A., Lawrence, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+protein+arginine+deiminase+activity+by+using+fluorescence+quenching:+multicolor+visualization+of+citrullination.">Monitoring of protein arginine deiminase activity by using fluorescence quenching: multicolor visualization of citrullination.</a> Angew. Chem. Int. Ed. Engl. 52, 2323-2325 (2013).</li><li>Jones, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+screening+of+a+haloacetamidine+containing+library+to+identify+PAD4+selective+inhibitors.">Synthesis and screening of a haloacetamidine containing library to identify PAD4 selective inhibitors.</a> ACS Chem. Biol. 7, 160-165 (2012).</li><li>Dreyton, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optimization and characterization of a pan protein arginine deiminase (PAD) inhibitor. , (2010).</li><li>Shimoyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deimination+stabilizes+histone+H2A/H2B+dimers+as+revealed+by+electrospray+ionization+mass+spectrometry.">Deimination stabilizes histone H2A/H2B dimers as revealed by electrospray ionization mass spectrometry.</a> J. Mass Spectrom. 45, 900-908 (2010).</li><li>Luo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+and+inactivators+of+protein+arginine+deiminase+4:+functional+and+structural+characterization.">Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization.</a> Biochemistry. 45, 11727-11736 (2006).</li></ol>

The authors have nothing to disclose.

Herein, a fluorescence based assay was successfully developed that monitors the activity of PAD4. The assay has proven to be incredibly robust in a number of high-throughput conditions and is also compatible with whole cell lysates37.
The affinity between the histone proteins and the DNA strands surrounding it loosens due the neutralization of the positive charge on the arginine side chain upon citrullination. It was envisioned that the neutralization of the arginine side-chain coul...

Um grande número de proteínas de mamífero são fortemente modificado pela acção de enzimas após a biossíntese de proteínas pelo ribossoma. Estas modificações pós-translacionais (PTMs) pode aumentar significativamente a diversidade funcional do proteoma mudando o tamanho, carga, estrutura, e estado de oligomerização (entre outras características) de proteínas 1-3. Como resultado, a variação na estrutura da proteína podem conduzir a consequências fisiológicas, tais como a degradação das proteínas, a diferenciação celular, a sinalização, a modulação da expressão do gene, e interacções proteína-proteína. Enquanto essas modificações são predominantes em uma grande porcentagem de todas as proteínas humanas, o terminal termina em proteínas histonas passam por um número anormalmente elevado de modificações covalentes 4. Proteínas histona são uma família de proteínas estruturais que facilitam a condensação de ADN genómico. Modificações covalentes das caudas das histonas não estruturados são realizadas e regulado por uma series de enzimas que podem catalisar a modificação covalente de resíduos (escritores), inverter as mesmas modificações (borrachas), e distinguem entre as mudanças que estão sendo impressas para as caudas das histonas (leitores) 5-7. Na verdade, a maioria dos PTMs conhecidos pode ser observado dentro deste segmento curto da histona, incluindo a metilação, fosforilação, acetilação, sumoilação, ubiquitinação, e citrulinação 8. Citrulinação envolve a conversão de peptidil-arginina para o t ARN não codificado peptidil-citrulina (Figura 1A). As proteínas, responsáveis ​​por esta cadeia lateral de neutralização, são membros da DAP (p eptide um eiminase rginine d) a família de proteínas, todas as quais são enzimas dependentes de cálcio 9,10.. Até à data, cinco membros da família PAD foram descritos (PAD1, PAD2, PAD3, PAD4, e PAD6). Cada membro desta família parece alvejar proteínas celulares distintas e também exibe uniperfis de distribuição de tecido Que. PAD4 é o único membro desta família de proteínas conhecida por estar localizada dentro do núcleo através de uma sequência de localização nuclear 11. Deste modo, tem sido demonstrado que deiminate um número de alvos nucleares, incluindo cadeias laterais de arginina sobre as caudas N-terminais das histonas H2A arginina resíduo 3 (H2R3), H3 (H3R2, H3R17, e H3R26) e H4 (H4R3) 12, 13. Enquanto cada uma das isozimas PAD têm funções fisiológicas específicas e críticos, PAD4 tem recebido bastante mais atenção devido ao seu papel numa série de processos humanos em ambas as células doentes e saudáveis. Recentemente, PAD4 mostrou ser um membro da rede transcricional pluripotência 14. Ambos os níveis de expressão PAD4 e atividade mostraram-se elevados durante a reprogramação e estatais chão estados pluripotentes em camundongos. Ao controlar a regulação de genes de células-tronco, PAD4 pode reter um papel crucial na eficiência de reprogramação celular. PAD4 também tem sido implicado naformação de armadilhas extracelulares de neutrófilos, o qual após a ligação de agentes patogénicos possibilitar a sua recarga do sistema. O hypercitrullination de proteínas histona por PAD4 induz descondensação da cromatina, o que serve como o material de base para o encapsulamento de bactérias patogénicas por a armadilha extracelular, assim, afastar infecções bacterianas 15,16. Além disso, foi encontrado PAD4 a desempenhar um papel activo num número de doenças humanas. Foi previamente mostrado que a expressão aberrante de PAD4 está associada com o aparecimento e gravidade da artrite reumatóide, a doença de Alzheimer, doença de Parkinson, e esclerose múltipla 17. Na verdade, a presença de anticorpos anti-citrulinados proteína é um dos biomarcadores de diagnóstico e de prognóstico mais fiáveis ​​e definitivas 18 de artrite reumatóide. Do mesmo modo, a actividade desregulada PAD4 foi recentemente observado num número de cancros humanos incluindo ovário, mama, pulmão, umad cânceres de esôfago 19-22. A ligação entre PAD4 e cancro tem sido mostrado para ser mediada através do oncogene ELK1 ou através da proteína supressora de tumor p53 22,23 e trabalho anterior sugeriu que PAD4 poderia ser um alvo terapêutico anti-cancro da novela 17,24,25. Como prova do princípio do estudo, a depleção de PAD4 através shRNA na linha celular de cancro colo-rectal HCT116 foi revelado para ser suficiente para induzir a apoptose e paragem do ciclo celular 26. Um inibidor irreversível PAD4 recentemente desenvolvido conduziu a uma redução de setenta por cento da massa do tumor em ratos 27. Muito notavelmente, a inibição PAD4 apareceu para actuar como uma terapia-alvo que resultou na morte selectiva de células cancerígenas, enquanto poupando células não transformadas. O uso de pequenas moléculas para desligar a função de PAD4 pode vir a ser uma nova e poderosa estratégia para atingir as células cancerosas ou para aumentar quimioterápicos de câncer existentes 28. Infelizmente, a potenda inibidor reversível PAD4 tem ainda a ser descoberto. Uma série de inibidores covalentes foram desenvolvidos usando o / alça imidine flúor cloro que imita o substrato arginina 27,29,30 e provaram ser ferramentas práticas para a compreensão do papel da PAD4 em ambas as células estaduais saudáveis ​​e doentes. No entanto, estas moléculas inibem a todos os PADs activos com potência similar. Portanto, a necessidade de um ensaio fácil, que relata sobre a actividade de PAD4 é crucial. Até à data, PAD4 ensaios foram descritos que vincular a liberação de amônia a partir da reação de uma leitura colorimétrica 31, utilizar um análogo do substrato chloroamidine fluorescente etiquetado para teste de polarização fluorescente de 32, contam com a reação assistida por ácido entre glioxal e citrulina 33, e par a actividade PAD4 a um passo de fluorescência dequenching 34. Destes, apenas a estratégia haloacetamidine modificador covalente tem provado ser compatíveis com scre de alto rendimentoplataformas ening 32,35,36. Nós descrevemos um ensaio baseado fluorescência fácil, que mede de forma confiável a atividade de PAD4. O ensaio, que exibe uma forte relação de sinal-para-ruído, a velocidade de análise, e robustez de medição, tem o potencial para descobrir um verdadeiramente potente e selectivo inibidor PAD4.

<table border="0" cellpadding="0" cellspacing="0" width="716">
	<tbody>
		<tr height="46">
			<td height="46" style="height:46px;width:192px;">Name of the Material/Equipment</td>
			<td style="width:123px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:267px;">Comments/ Description</td>
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		<tr height="66">
			<td height="66" style="height:66px;width:192px;">LB Broth, Miller (LURIA-BERTANI)</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:135px;">J106-2KG</td>
			<td style="width:267px;"><a href="http://www.amresco-inc.com/">http://www.amresco-inc.com</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:192px;">Ampicillin Trihydrate (Off-white Powder), Fisher BioReagents</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td>BP902-25&#160;</td>
			<td style="width:267px;"><a href="http://www.fishersci.com/">http://www.fishersci.com</a></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:192px;">Isopropylthiagalactonse (IPTG)</td>
			<td style="width:123px;">Life Technogolies</td>
			<td>15529-019</td>
			<td style="width:267px;"><a href="http://www.lifetechnologies.com/">http://www.lifetechnologies.com</a></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:192px;">All Buffer Salts&#160;</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Can purchase from Fisher Scieintifc; VWR; Acros; Sigma-Aldrich; etc.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:192px;">Protino Glutathione (GSH) agarose&#160;</td>
			<td style="width:123px;">Machery-Nagel</td>
			<td style="width:135px;">745500.10</td>
			<td style="width:267px;">Store at 4 &#176;C; http://www.mn-net.com/</td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:192px;">Z-&#8203;Arg-&#8203;Arg-&#8203;7-&#8203;amido-&#8203;4-&#8203;methylcoumarin hydrochloride (Zcoum)</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td>C5429</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:192px;">Chloroamidine&#160;</td>
			<td style="width:123px;">Cayman Chemical&#160;</td>
			<td style="width:135px;">10599</td>
			<td style="width:267px;">Store at -20 &#176;C; https://www.caymanchem.com/app/template/Home.vm</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:192px;">Tris(2-&#8203;carboxyethyl)&#8203;phosphine hydrochloride (TCEP HCl)</td>
			<td style="width:123px;">Thermo Scientifc</td>
			<td style="width:135px;">20490</td>
			<td style="width:267px;"><a href="http://www.piercenet.com/">http://www.piercenet.com</a></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:192px;">Triton X-100</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:135px;">X100-100ML</td>
			<td style="width:267px;"><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:192px;">Corning/Nunclon MicroWell plates (96 and 384)</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;">Purchase from Corning; Sigma-Aldrich</td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:192px;">Tecan Infinite 200 / Tecan i-control microplate reader software</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.tecan.com/">www.tecan.com</a></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:192px;">Europium Ex. 340/40 Em. 475/15 filter</td>
			<td style="width:123px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td style="width:267px;"><a href="http://www.perkinelmer.com/">http://www.perkinelmer.com</a></td>
		</tr>
	</tbody>
</table>

fluorescence-based monitoring of pad4 activity via a pro-fluorescence substrate analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

Inicialmente, foi demonstrado que ZRcoum pode informar sobre a actividade de PAD4 num formato de placa de 96 poços. Os poços foram incubados com o substrato ZRcoum e na presença / ausência de PAD4. Após um período de incubação de 45 min a 37 ° C, a fluorescência foi medida com um 340/40 - 475/15 nm filtro (Figura 2A). Como esperado, os níveis permaneceram baixos de fluorescência como o fluoróforo dentro ZRcoum (ou a citrulina ZRcoum...

Watch this Scientific Journal Video about Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog at JoVE.com

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Monitoramento baseado em fluorescência de PAD4 Atividade através de um substrato Analog Pro-fluorescência

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

fluorescence-based-monitoring-pad4-activity-via-pro-fluorescence

Research

JoVE Journal

Chemistry

9.8K visualizações.  Lehigh University. PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Vídeo: Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

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

Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.

This article describes a rapid supercritical extraction method for fabricating silica aerogels. By utilizing a confined mold and hydraulic hot press, monolithic aerogels can be made in eight hours or less.

Preparação de sílica aerogel Monólitos através de um Método de extracção rápida supercrítico

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

Monitoramento em tempo real de Reações realizada utilizando processamento de fluxo contínuo: A Preparação de 3-Acetylcoumarin como Exemplo

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

Medição simultânea de HDAC1 e atividade HDAC6 em HeLa células usando UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

Fabricação e teste de catalítico Aerogels preparado através da rápida extração supercrítica

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Síntese de nanofios de Au substrato ligados através de um mecanismo de crescimento ativo de superfície

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

A Two-Step Protocol for Umpolung Functionalization of Ketones Via Enolonium Species

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. Recently, we have developed a two-step strategy for ketone enolate umpolung that has enabled the development of methods for chlorination, azidation, and amination using azoles. In addition, we have developed C-C bond&#8211;forming arylation and allylation reactions. At the heart of these methods is the preparation of the intermediate and highly reactive enolonium species prior to addition of a reactive nucleophile. This strategy is thus reminiscent of the preparation and use of metal enolates in classical synthetic chemistry. This strategy allows the use of nucleophiles that would otherwise be incompatible with the strongly oxidizing hypervalent iodine reagents. In this paper we present a detailed protocol for chlorination, azidation, N-heteroarylation, arylation, and allylation. The products include motifs prevalent in medicinally active products. This article will greatly assist others in using these methods.

A two step one-pot protocol for the umpolung of ketone enolates to enolonium species and addition of a nucleophile to the &#945;-position is described. Nucleophiles include chloride, azide, azoles, allyl-silanes, and aromatic compounds.

Um Protocolo em Duas Etapas para Funcionalização de Umpolung de Cetonas via Espécies de Enolonium

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

Síntese de Ésteres Via Esterificação de Steglich Mais Sustentável em Acetonitrila

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

Ensaios de atividade com base em RMN para determinação da inibição de compostos, valores de IC50 , atividade Artifactual e atividade de células inteiras de Ribohidrolases nucleósidos

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Técnicas de espectrometria de massa de mobilidade de íons para determinação da estrutura e mecanismos de reconhecimento de íons metálicos e atividade redox de oligopeptídeos de ligação metálica