Name: label-free quantitative proteomics workflow for discovery-driven host-pathogen interactions
Uploaded: 2020-10-20T14:30:00
Description: Watch this Scientific Journal Video about Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions at JoVE.com

Os autores agradecem ao Dr. Jonathan Krieger da Bioinformatics Solutions Inc. por operar o espectrômetro de massa para experimentos representativos, bem como membros do grupo Geddes-McAlister por sua assistência com configuração experimental e feedback de manuscritos. Os autores reconhecem o apoio ao financiamento, em parte, da Fundação de Pesquisa Banting – The Jarislowsky Fellowship Discovery Award, New Frontiers Research Fund – Exploration (NFRFE-2019-00425), e da Canadian Foundation for Innovation (JELF 38798) para J.G.M., bem como bolsa de pós-graduação da NSERC Canada – Mestrado e Bolsa de Pós-Graduação de Ontário para B.B., e Bolsa de Pós-Graduação Rainha Elizabeth II em Ciência e Tecnologia para A.S..

Uma linha imortalizada de macrófagos derivados de camundongos BALB/c foram usados para o seguinte protocolo aprovado pelo Protocolo de Utilização animal da Universidade de Guelph 4193. Notavelmente, outras cepas de camundongos ou outras fontes de células imortalizadas podem ser aplicadas ao protocolo delineado com testes suficientes para otimizar os parâmetros detalhados. O protocolo a seguir navegará pelas etapas que começam com um frasco congelado de células macrófagos. As células são armazenadas em 10% FBS ...

<ol>
	<li>Bongomin, F., Gago, S., Oladele, R., Denning, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+Multi-National+Prevalence+of+Fungal+Diseases-Estimate+Precision.">Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision.</a> Journal of Fungi. , (2017).</li><li>Tugume, L., 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=HIV-Associated+cryptococcal+meningitis+occurring+at+relatively+higher+CD4+counts.">HIV-Associated cryptococcal meningitis occurring at relatively higher CD4 counts.</a> Journal of Infectious Diseases. 219 (6), 877-883 (2019).</li><li>Rajasingham, R., 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=Global+burden+of+disease+of+HIV-associated+cryptococcal+meningitis:+an+updated+analysis.">Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis.</a> The Lancet Infectious Diseases. , (2017).</li><li>Perfect, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antifungal+pipeline:+A+reality+check.">The antifungal pipeline: A reality check.</a> Nature Reviews Drug Discovery. , (2017).</li><li>Bermas, A., Geddes-McAlister, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combatting+the+evolution+of+anti-fungal+resistance+in+Cryptococcus+neoformans.">Combatting the evolution of anti-fungal resistance in Cryptococcus neoformans.</a> Molecular Microbiology. , 1-14 (2020).</li><li>Geddes-McAlister, J., Shapiro, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+pathogens,+new+tricks:+Emerging,+drug-resistant+fungal+pathogens+and+future+prospects+for+antifungal+therapeutics.">New pathogens, new tricks: Emerging, drug-resistant fungal pathogens and future prospects for antifungal therapeutics.</a> Annals of the New York Academy of Sciences. , (2018).</li><li>Ball, B., Bermas, A., Carruthers-Lay, D., Geddes-McAlister, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometry-Based+Proteomics+of+Fungal+Pathogenesis,+Host-Fungal+Interactions,+and+Antifungal+Development.">Mass Spectrometry-Based Proteomics of Fungal Pathogenesis, Host-Fungal Interactions, and Antifungal Development.</a> Journal of Fungi. , (2019).</li><li>Sukumaran, A., 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=Decoding+communication+patterns+of+the+innate+immune+system+by+quantitative+proteomics.">Decoding communication patterns of the innate immune system by quantitative proteomics.</a> J Leukocyte Biol. , (2019).</li><li>Salas, D., Stacey, R. G., Akinlaja, M., Foster, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+interactomics:+Considerations+for+the+use+of+co-elution+to+measure+protein+interaction+networks.">Next-generation interactomics: Considerations for the use of co-elution to measure protein interaction networks.</a> Molecular and Cellular Proteomics. , (2020).</li><li>Aebersold, R., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-spectrometric+exploration+of+proteome+structure+and+function.">Mass-spectrometric exploration of proteome structure and function.</a> Nature. 537 (7620), 347-355 (2016).</li><li>Mann, M., Kulak, N. A., Nagaraj, N., Cox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Coming+Age+of+Complete,+Accurate,+and+Ubiquitous+Proteomes.">The Coming Age of Complete, Accurate, and Ubiquitous Proteomes.</a> Molecular Cell. 49 (4), 583-590 (2013).</li><li>Cox, 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=Accurate+Proteome-wide+Label-free+Quantification+by+Delayed+Normalization+and+Maximal+Peptide+Ratio+Extraction,+Termed+MaxLFQ.">Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ.</a> Molecular &amp; Cellular Proteomics. 13 (9), 2513-2526 (2014).</li><li>Ankney, J. A., Muneer, A., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+and+Absolute+Quantitation+in+Mass+Spectrometry-Based+Proteomics.">Relative and Absolute Quantitation in Mass Spectrometry-Based Proteomics.</a> Annual Review of Analytical Chemistry. , (2018).</li><li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MaxQuant+enables+high+peptide+identification+rates,+individualized+p.p.b.-range+mass+accuracies+and+proteome-wide+protein+quantification.">MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.</a> Nature Biotechnology. 26 (12), 1367-1372 (2008).</li><li>Tyanova, 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=The+Perseus+computational+platform+for+comprehensive+analysis+of+(prote)omics+data.">The Perseus computational platform for comprehensive analysis of (prote)omics data.</a> Nature Methods. 13 (9), 731-740 (2016).</li><li>Cox, 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=Andromeda:+A+peptide+search+engine+integrated+into+the+MaxQuant+environment.">Andromeda: A peptide search engine integrated into the MaxQuant environment.</a> Journal of Proteome Research. , (2011).</li><li>Zhong, Z., Pirofski, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opsonization+of+Cryptococcus+neoformans+by+human+anticryptococcal+glucuronoxylomannan+antibodies.">Opsonization of Cryptococcus neoformans by human anticryptococcal glucuronoxylomannan antibodies.</a> Infection and Immunity. , (1996).</li><li>Nicola, A. M., 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=Macrophage+autophagy+in+immunity+to+Cryptococcus+neoformans+and+Candida+albicans.">Macrophage autophagy in immunity to Cryptococcus neoformans and Candida albicans.</a> Infection and Immunity. , (2012).</li><li>Ball, B., Geddes-McAlister, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Proteomic+Profiling+of+Cryptococcus+neoformans.">Quantitative Proteomic Profiling of Cryptococcus neoformans.</a> Current Protocols in Microbiology. , (2019).</li><li>Geddes-McAlister, J., Gadjeva, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectromerty-based+quantitative+proteomics+of+murine-derived+polymorphonuclear+neutrophils.">Mass spectromerty-based quantitative proteomics of murine-derived polymorphonuclear neutrophils.</a> Current Protocols in Immunology. , (2019).</li><li>Geddes, J. M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretome+profiling+of+Cryptococcus+neoformans+reveals+regulation+of+a+subset+of+virulence-associated+proteins+and+potential+biomarkers+by+protein+kinase+A.">Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A.</a> BMC Microbiology. , (2015).</li><li>Geddes, J. M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+protein+kinase+a-regulated+proteome+of+Cryptococcus+neoformans+identifies+a+role+for+the+ubiquitin-proteasome+pathway+in+capsule+formation.">Analysis of the protein kinase a-regulated proteome of Cryptococcus neoformans identifies a role for the ubiquitin-proteasome pathway in capsule formation.</a> mBio. 7 (1), 1-15 (2016).</li><li>Al Shweiki, M. H. D. R., 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=Assessment+of+Label-Free+Quantification+in+Discovery+Proteomics+and+Impact+of+Technological+Factors+and+Natural+Variability+of+Protein+Abundance.">Assessment of Label-Free Quantification in Discovery Proteomics and Impact of Technological Factors and Natural Variability of Protein Abundance.</a> Journal of Proteome Research. , (2017).</li><li>Ong, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Isotope+Labeling+by+Amino+Acids+in+Cell+Culture,+SILAC,+as+a+Simple+and+Accurate+Approach+to+Expression+Proteomics.">Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics.</a> Molecular &amp; Cellular Proteomics. 1 (5), 376-386 (2002).</li><li>Thompson, A., 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=Tandem+mass+tags:+A+novel+quantification+strategy+for+comparative+analysis+of+complex+protein+mixtures+by+MS/MS.">Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.</a> Analytical Chemistry. 75 (8), 1895-1904 (2003).</li><li>Borner, G. H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organellar+Maps+Through+Proteomic+Profiling+-+A+Conceptual+Guide.">Organellar Maps Through Proteomic Profiling - A Conceptual Guide.</a> Molecular &amp; Cellular Proteomics. , (2020).</li><li>Gingras, A. C., Abe, K. T., Raught, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+know+the+neighborhood:+using+proximity-dependent+biotinylation+to+characterize+protein+complexes+and+map+organelles.">Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles.</a> Current Opinion in Chemical Biology. , (2019).</li><li>Kugadas, A., 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=Frontline+Science:+Employing+enzymatic+treatment+options+for+management+of+ocular+biofilm-based+infections.">Frontline Science: Employing enzymatic treatment options for management of ocular biofilm-based infections.</a> Journal of Leukocyte Biology. , (2019).</li><li>Yeung, J., Gadjeva, M., Geddes-McAlister, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Quantitative+Proteomics+Distinguishes+General+and+Site-Specific+Host+Responses+to+Pseudomonas+aeruginosa+Infection+at+the+Ocular+Surface.">Label-Free Quantitative Proteomics Distinguishes General and Site-Specific Host Responses to Pseudomonas aeruginosa Infection at the Ocular Surface.</a> Proteomics. , (2020).</li><li>Yeung, J., Lamb, J., Krieger, J. R., Gadjeva, M., Geddes-McAlister, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Proteomic+Profiling+ofMurine+Ocular+Tissue+and+theExtracellular+Environment.">Quantitative Proteomic Profiling ofMurine Ocular Tissue and theExtracellular Environment.</a> Current Protocols in Mouse Biology. 10 (83), (2020).</li></ol>

As etapas críticas do protocolo incluem a preparação de células macrófagos e a coleta de amostras de co-cultura para processamento de proteínas com interrupção mínima das células. É importante realizar etapas de lavagem, inoculação e remoção de células macrófagos aderentes suavemente e cuidadosamente para evitar a lise desnecessária das células antes da coleta. Estabelecer o MOI correto para o experimento também é fundamental, pois a inoculação com um MOI excessivamente alto pode causar morte rápi...

A prevalência de infecções fúngicas invasivas está aumentando muito e está correlacionada com taxas inaceitáveis de mortalidade elevadas, mais comumente relatadas em indivíduos com predisposições imunodeficientes1. Cryptococcus neoformans é um notória patógeno fúngico oportunista capaz de sobrevivência intracelular dentro de células macrófagos hospedeiras. Intervenção antifúngica inadequada resulta em disseminação fúngica e manifestações de risco de vida de meningite criptocócica e meningoencefalite2,3. O aumento global do status imunocomprometido exigiu um aumento paralelo no uso de agentes antifúngicos, nos quais muitas espécies fúngicas, incluindo C. neoformans, têm evoluído cada vez mais resistência para4,5,6. Portanto, é imprescindível implementar tecnologias robustas e eficientes para responder a questões biológicas vitais sobre resposta à defesa do hospedeiro e patogênese microbiana.
A nova era do avanço tecnológico na espectrometria de massa (MS), incluindo a geração de poderosos gasodutos computacionais e bioinforáticos, fornece a base para uma visão integrativa para análise em larga escala da pesquisa hospedeira-patógeno7,8. A análise proteômica convencional orientada pela patogênese geralmente perfila a visão da infecção da perspectiva hospedeira ou patógena, incluindo metodologias abrangentes como perfil de correlação de proteínas, cromatografia de afinidade combinada com proteômica e interactomics9. As investigações sobre a virulência de patógenos perigosos em um sistema hospedeiro são de imensa importância clínica; no entanto, a aplicação de uma análise de dupla perspectiva em um único experimento foi anteriormente considerada inatingível. Por exemplo, a perspectiva do patógeno em relação à infecção é frequentemente sobrecarregada por proteínas hospedeiras altamente abundantes, resultando em sensibilidade reduzida para a detecção de proteínas fúngicas de baixa abundantes7. Além disso, a alta complexidade amostral convida muitos alvos a investigar em um único sistema experimental e se mostra desafiador para elucidar mecanismos de ação para uma proteína patógena específica.
A proteômica de baixo para cima é uma técnica popular de MS que permite a preparação de amostras gerenciáveis, na qual os peptídeos são gerados pela digestão enzimática específica da sequência seguida pela separação, identificação e quantificação da cromatografia líquida pela MS10,11. Aqui, apresentamos um método demonstrando uma estratégia de aquisição dependente de dados com o objetivo de alcançar uma cobertura imparcial de um proteome baseado em infecção ou "infectome". Especificamente, a quantificação sem rótulos (LFQ) libera a dependência de rótulos químicos ou metabólicos para identificação robusta e precisa de alterações no nível de proteínas em múltiplos proteomes, reduzindo as etapas de manuseio e processamento de amostras12,13. Esta aplicação universal interroga proteínas produzidas em um dado momento dentro de uma célula independente de qualquer produção de proteína esperada; assim, novos insights podem ser descobertos que são críticos para a infecção.
O fluxo de trabalho descrito aqui é otimizado para explorar mudanças no nível de proteína de C. neoformans durante condições de imitação de infecções com células imunes hospedeiras(Figura 1). Em vez de depender do isolamento e separação dos tipos celulares, essa abordagem extrai o hospedeiro e o proteome patógeno juntos, e utiliza a separação bioinformática usando dois bancos de dados específicos do organismo para distinguir a produção de proteínas específicas das espécies. Este método oferece vantagens para um número ilimitado de amostras a serem processadas sem as etapas de preparação extra dispendias necessárias em estudos de rotulagem ou fracionamento baseados em isótopos. Além disso, este fluxo de trabalho suporta protocolos otimizados de extração de proteínas transferíveis para uma ampla gama de patógenos fúngicos e bacterianos capazes de atingir e infectar células imunes hospedeiras. No geral, este protocolo descreve as etapas para concluir uma extração de proteína imparcial e processamento de amostras para MS de alta resolução, seguida de dados e análise estatística, capaz de fornecer uma riqueza de conhecimento de proteínas fúngicas significativas para infecção combinada com o perfil abrangente da resposta à defesa do hospedeiro.

<table>
	<tbody>
		<tr>
			<td>100 mM Tris-HCl, pH 8.5</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Maintain at 4&#176;C</td>
		</tr>
		<tr>
			<td>60 x 15 mm Dish, Nunclon Delta</td>
			<td>ThermoFisher Scientific</td>
			<td>174888</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>ThermoFisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, MS grade</td>
			<td>Pierce</td>
			<td>TS-51101</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>1099510001</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma Aldrich</td>
			<td>34850-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate (ABC)</td>
			<td>ThermoFisher Scientific</td>
			<td>A643-500</td>
			<td>Prepare a stock 50 mM ABC solution, stable at room temperature for up to one month.</td>
		</tr>
		<tr>
			<td>Bel-Art&#8482; HiFlow Vacuum Aspirator Collection System</td>
			<td>Fisher Scientific</td>
			<td>13-717-300</td>
			<td>Not essential, serological pipettes can be used to remove media.</td>
		</tr>
		<tr>
			<td>C18 resin</td>
			<td>3M Empore</td>
			<td>3M2215</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scrapers</td>
			<td>VWR</td>
			<td>10062-906</td>
			<td>Not essential, other methods to release macrophage cells can be used.</td>
		</tr>
		<tr>
			<td>Centrifugal vaccuum concentrator</td>
			<td>Eppendorf</td>
			<td>07-748-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Filtration Unit</td>
			<td>VWR</td>
			<td>10040-436</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical falcon tubes (15 mL)</td>
			<td>Fisher Scientific</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>ThermoFisher Scientific</td>
			<td>AMQAX1000</td>
			<td>Not essential, haemocytometer can be used as an alternative.</td>
		</tr>
		<tr>
			<td>CytoTox 96 Non-Radioactive Cytotoxicity Assay</td>
			<td>Promega</td>
			<td>G1780</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>ThermoFisher Scientific</td>
			<td>R0861</td>
			<td>Prepare bulk stock solution of 1 M DTT, flash frozen and stored at -20 &#176;C until use. Discard after each use (do not freeze-thaw repeatedly).</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>10566016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>12483020</td>
			<td>Heat inactivate by incubating at 60&#176;C for 30 minutes. Prepare 50 ml aliquots and flash freeze. Thaw prior to media preparation</td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td>VWR</td>
			<td>15170-208</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20&#176;C until use. Discard after each use (do not freeze-thaw repeatedly).</td>
		</tr>
		<tr>
			<td>High-performance liquid chromatography system</td>
			<td>ThermoFisher Scientific</td>
			<td>LC140</td>
			<td>Gradient length is based on sample complexity, recommended 120 min gradient for infectome samples.</td>
		</tr>
		<tr>
			<td>High-resolution mass spectrometer</td>
			<td>ThermoFisher Scientific</td>
			<td>726042</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma Aldrich</td>
			<td>I6125</td>
			<td>Prepare 0.55 M bulk stock solution, flash frozen, store at -20&#176;C until use. Discard after each use (do not freeze-thaw repeatedly).</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>25030081</td>
			<td>Can be aliquot and frozen for storage. Thaw prior to media preparation.</td>
		</tr>
		<tr>
			<td>LoBind Microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>13-698-794</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>https://maxquant.org/</td>
			<td></td>
			<td>MaxQuant is a public platform that offers tutorials, such as the MaxQuant Summer School, outlining the computational analysis steps of large MS data sets</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>13864457</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin : Streptomycin 10k/10k</td>
			<td>VWR</td>
			<td>CA12001-692</td>
			<td>Can be aliquot and frozen for storage. Thaw prior to media preparation.</td>
		</tr>
		<tr>
			<td>Peptide separation columns</td>
			<td>ThermoFisher Scientific</td>
			<td>ES803</td>
			<td></td>
		</tr>
		<tr>
			<td>Perseus Software</td>
			<td>http://maxquant.net/perseus/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>VWR</td>
			<td>CA12001-676</td>
			<td>Puchase not required. PBS can also be prepared but sterile filteration must be performed before use.</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, Disposable Serological (10 mL)</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, Disposable Serological (25 mL) Basix</td>
			<td>Fisher Scientific</td>
			<td>14955235</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe sonciator</td>
			<td>ThermoFisher Scientific</td>
			<td>100-132-894</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablet</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>ThermoFisher Scientific</td>
			<td>28364</td>
			<td>20% (w/v)</td>
		</tr>
		<tr>
			<td>Spectrophotometer (Nanodrop)</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>STAGE tipping centrifuge</td>
			<td>Sonation</td>
			<td>STC-V2</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Shaker</td>
			<td>VWR</td>
			<td>NO89232-908</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>ThermoFisher Scientific</td>
			<td>85183</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C protease mix, MS grade</td>
			<td>Pierce</td>
			<td>A40007</td>
			<td>Maintain at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>Bransonic</td>
			<td>A89375-450</td>
			<td>Stored in cold room (4C)</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma Aldrich</td>
			<td>U1250-1KG</td>
			<td>Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20 &#176;C until use. Discard after each use (do not freeze-thaw repeatedly).</td>
		</tr>
		<tr>
			<td>Yeast-extract peptone dextrose broth</td>
			<td>BD Difco</td>
			<td>BM20</td>
			<td></td>
		</tr>
	</tbody>
</table>

label-free quantitative proteomics workflow for discovery-driven host-pathogen interactions

As realizações tecnológicas da proteômica quantitativa baseada em espectrometria de massa (MS) abrem muitas vias desconhecidas para analisar o proteome global de um organismo em condições variadas. Esta poderosa estratégia aplicada às interações de patógenos microbianos com o hospedeiro desejado caracteriza de forma abrangente ambas as perspectivas para a infecção. Aqui, o fluxo de trabalho descreve a quantificação sem rótulos (LFQ) do infectome de Cryptococcus neoformans, um patógeno intracelular facultativo fúngico que é o agente causador da doença mortal criptococcose, na presença de células macrófagos imortalizadas. O protocolo detalha as técnicas adequadas de preparação de proteínas para células patógenas e mamíferos dentro de um único experimento, resultando em submissão adequada de peptídeos para análise de cromatografia líquida (LC)-MS/MS. A natureza genérica de alto rendimento do LFQ permite uma ampla gama dinâmica de identificação e quantificação de proteínas, bem como transferência para qualquer configuração de infecção por hospedeiro-patógeno, mantendo extrema sensibilidade. O método é otimizado para catalogar perfis extensos e imparcial de abundância de proteínas de um patógeno em condições de imitação de infecções. Especificamente, o método aqui demonstrado fornece informações essenciais sobre a patogênese C. neoformans, como a produção de proteínas necessárias para a virulência e identifica proteínas críticas de hospedeiro que respondem à invasão microbiana.

O protocolo descrito acima permite a identificação e quantificação de proteínas derivadas tanto do patógeno fúngico, C. neoformans, quanto do hospedeiro, células macrófagos, em um único experimento. Seguindo a cocultura, as células são coletadas e processadas juntas e bioinformáticas separadas com base em perfis de peptídeos específicos para cada espécie. Esta é uma abordagem poderosa para definir a interação da relação hospedeiro-patógeno durante a infecção. O número de proteínas ident...

Watch this Scientific Journal Video about Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions at JoVE.com

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

Aqui, apresentamos um protocolo para traçar o perfil entre hospedeiro e patógeno durante a infecção por proteômica baseada em espectrometria de massa. Este protocolo usa quantificação livre de rótulos para medir mudanças na abundância de proteínas tanto do hospedeiro (por exemplo, macrófagos) quanto de patógenos (por exemplo, Cryptococcus neoformans) em um único experimento.

Fluxo de trabalho de proteômica quantitativa sem rótulo para interações de patógenos de host-pathogen orientadas para a descoberta

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Our protocol investigates fungal infection from both the host and pathogen perspectives to identify interactions between the biological systems that are critical for a disease. We can detect both fungal and host proteins in a single experiment and identify fungal proteins only produced during infection, representing novel infection-associated proteins. Although we focus on treating fungal infections by uncovering novel anti-virulent strategies for treatment, our proteomics and bioinformatics pipeline is universal and could be applied to many biological systems.Our approach lays the foundation for studies of different pathogens and numerous immune responses and can be used to provide new insights into the relationship between cryptococcus and the innate immune system. The macrophage response to pathogens and detecting enough fungal proteins are important. Therefore testing and optimizing MOIs and time points for each species are recommended.As macrophage and fungal cell culture can be challenging, knowing what to expect after coculture is important. Visualization provides the researcher with confidence in implementing the procedure. To prepare fungal cells for a macrophage infection, collect and centrifuge C neoformans cells from a mid long phase culture, followed by three gentle washes with one milliliter of room temperature PBS under the same centrifuge conditions.After the last wash, resuspend the fungal cells at a 1.2 times 10 to the eighth cells per milliliter of antibiotic free cell culture, medium concentration and add one milliliter of fungal cell suspension to each of four wells of a 70%to 80%confluent six well macrophage culture plate. Then, place the plate in the cell culture incubator for three hours. Carefully tilt the plate to allow each well to be gently washed three times with one milliliter of PBS per well per wash.To measure the infection proficiency after the last wash, add one milliliter of antibiotic free medium to each well of the six well plate, and place the plate in the cell culture incubator. At the appropriate experimental time points, collect the supernatant from each well and measure the amount of LDH in each well according to standard protocols. At the same time points, lyse the uninfected macrophage cells to determine the value for maximum cytotoxicity.The cytotoxicity can then be calculated using the formula as indicated. For uninfected macrophage collection and co-culture, add one milliliter of cold PBS to each well of uninfected macrophages. After one minute, gently tap the plate to detach the cells from the bottoms of the wells and pool the cell suspensions into a single 15 milliliter tube.Collect the cells by centrifugation and completely remove the supernatant to allow immediate flash freezing of the pellet in liquid nitrogen for minus 80 degree storage. Principal component analysis of the process to datasets reveals that, as expected, the largest component of separation among the data is infected versus non-infected samples. The second distinguishing feature of the samples is their biological variability.Combining a Pearson correlation with hierarchical clustering by Euclidean distance shows a distinct clustering of infected versus non-infected samples with a replicate reproducibility ranging from 95%to 96%Students'T test corrected for multiple hypothesis testing using a Benjamini-Hochberg False Discovery Rate can be performed to identify proteins with significant differences in abundance during infection compared to non-infected controls. In this analysis, 117 host proteins demonstrating a significant change in expression upon infection were identified. Notably, significant increases in the abundance of fungal proteins, as expected during infection, were also observed.It is important to handle the macrophages gently during coculturing, washing, and sample collection. By defining how the pathogen adapts to the host and how the host defends itself from infection, new insights are acquired into the fields of microbiology and immunology.

Research

JoVE Journal

Immunology and Infection

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Uso de uma armadilha óptica de Estudos de Interações Hospedeiro-Patógeno para imagens de células dinâmica ao vivo

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Imunologia e Infecção

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Utilização do Modelo EpiAirway para caracterizar a longo prazo Interações Hospedeiro-Patógeno

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Uma abordagem comparativa para caracterizar a paisagem de Patógeno-Hospedeiro interações proteína-proteína

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

Detecção de Leucócitos Humanos Antigen Biomarkers no cancro da mama Utilizando tecnologia de biossensores livre-Label

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

A electroporação de efectores bacterianos funcionais em células de mamíferos

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Avaliação da eficácia e toxicidade de RNAs Segmentação HIV-1 Produção para uso em Gene ou Quimioterapia

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Um novo método para a análise qualitativa Multi-escala de Bacteriana biofilmes em filamentosa fúngica Colonies Usando Confocal e Microscopia Eletrônica

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Ex Vivo Infecção da Mucosa Genital humano tecido linfoide e fêmea com vírus de imunodeficiência humana 1 e Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Livre de rótulo neutrófilo enriquecimento de secreção do paciente-derivado das vias aéreas usando microfluídica inercial de circuito fechado

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

Livre etiqueta de identificação dos subtipos de linfócito utilizando imagens tridimensionais fase quantitativa e aprendizagem de máquina

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...