The authors thank Dr. Jonathan Krieger of Bioinformatics Solutions Inc. for operating the mass spectrometer for representative experiments, as well as members of the Geddes-McAlister group for their assistance with experimental set-up and manuscript feedback. The authors acknowledge funding support, in part, from the Banting Research Foundation &#8211; The Jarislowsky Fellowship Discovery Award, New Frontiers Research Fund &#8211; Exploration (NFRFE-2019-00425), and the Canadian Foundation for Innovation (JELF 38798) for J.G.M., as well as NSERC Canada Graduate Scholarship &#8211; Masters and Ontario Graduate Scholarship for B.B., and Queen Elizabeth II Graduate Scholarship in Science and Technology for A.S..

An immortalized line of macrophages derived from BALB/c mice were used for the following protocol approved by the University of Guelph Animal Utilization Protocol 4193. Notably, other strains of mice or other sources of immortalized cells can be applied to the outlined protocol with sufficient testing to optimize the detailed parameters. The following protocol will navigate the steps beginning with a frozen vial of macrophage cells. Cells are stored in 10% FBS (fetal bovine serum), 1% L-glutamine and 5% Pen/Strep (Penici...

<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. 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Critical steps in the protocol include preparation of macrophage cells and collection of co-culture samples for protein processing with minimal disruption to the cells. It is important to perform steps of washing, inoculating, and removing adherent macrophage cells gently and carefully to prevent unnecessary lysis of cells prior to collection. Establishing the correct MOI for the experiment is also critical as inoculating with an excessively high MOI can cause rapid macrophage cell death and difficulty in collecting and ...

The prevalence of invasive fungal infections is vastly increasing and is correlated with unacceptably high mortality rates, most commonly reported in individuals with immunodeficient predispositions1. Cryptococcus neoformans is a notorious opportunistic fungal pathogen capable of intracellular survival within host macrophage cells. Inadequate antifungal intervention results in fungal dissemination and life-threatening manifestations of cryptococcal meningitis and meningoencephalitis2,3. The global increase in immunocompromised status has demanded a parallel increase in the use of antifungal agents, in which many fungal species, including C. neoformans, have increasingly evolved resistance towards4,5,6. Therefore, it is imperative to implement robust and efficient technologies to answer vital biological questions regarding host defence response and microbial pathogenesis.
The new age of technological advancement in mass spectrometry (MS), including the generation of powerful computational and bioinformatic pipelines, provides the foundation for an integrative vision for large-scale analysis of host-pathogen research7,8. Conventional pathogenesis-driven proteomic analysis commonly profiles the view of infection from either the host or pathogen perspective, including comprehensive methodologies such as protein correlation profiling, affinity chromatography combined with proteomics, and interactomics9. Investigations into the virulence of dangerous pathogens in a host system are of immense clinical importance; however, the application of a dual perspective analysis in a single experiment was formerly considered unattainable. For example, the pathogen&#8217;s perspective towards infection is often overwhelmed by highly abundant host proteins resulting in reduced sensitivity for the detection of low-abundant fungal proteins7. Furthermore, the high sample complexity invites many targets to investigate in a single experimental system and proves challenging to elucidate mechanisms of action for a specific pathogen protein.
Bottom-up proteomics is a popular MS technique that enables manageable sample preparation, in which peptides are generated by sequence-specific enzymatic digestion followed by liquid chromatography separation, identification, and quantification by MS10,11. Here, we present a method demonstrating a data-dependent acquisition strategy purposed to achieve an unbiased coverage of an infection-based proteome or &#8216;infectome&#8217;. Specifically, label-free quantification (LFQ) sheds the dependence on chemical or metabolic labels for robust and accurate identification of protein level changes across multiple proteomes, reducing sample handling and processing steps12,13. This universal application interrogates produced proteins at a given moment within a cell independent of any expected protein production; thus, novel insights may be discovered that are critical to infection.
The workflow described herein is optimized to explore protein level changes of C. neoformans during infection-mimicking conditions with host immune cells (Figure 1). Rather than relying on the isolation and separation of cell types, this approach extracts the host and pathogen proteome together, and utilizes bioinformatic separation using two organism-specific databases to distinguish species-specific protein production. This method offers advantages for an unlimited number of samples to be processed without the extra costly preparation steps necessary in isotope-based labelling studies or fractionation. Furthermore, this workflow supports optimized protein extraction protocols transferable to a wide range of fungal and bacterial pathogens capable of targeting and infecting host immune cells. Overall, this protocol outlines the steps to complete an unbiased protein extraction and sample processing for high-resolution MS, followed by data and statistical analysis, capable of providing a wealth of knowledge of fungal proteins significant for infection combined with comprehensive profiling of the host defense response.

<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

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

The protocol outlined above enables identification and quantification of proteins derived from both the fungal pathogen, C. neoformans, and the host, macrophage cells, in a single experiment. Following co-culture, cells are collected and processed together and bioinformatically separated based on peptide profiles specific to each species. This is a powerful approach for defining the interplay of the host-pathogen relationship during infection. The number of proteins identified from the experiment depends on the ...

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

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

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.

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

Research

JoVE Journal

Immunology and Infection

6.3K ビュー.  University of Guelph. 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 ...

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

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single...

a lactate dehydrogenase release assay to detect cryptococcus neoformans infection in macrophages

In this video, we demonstrate a cytotoxicity assay to assess Cryptococcus neoformans, a pathogenic fungus, infection in macrophages. The cytotoxicity is determined by the release of cytoplasmic lactate dehydrogenase enzyme due to macrophage lysis caused by Cryptococcus neoformans...

A Lactate Dehydrogenase Release Assay to Detect Cryptococcus neoformans Infection in Macrophages

label-free immunoprecipitation mass spectrometry workflow for large-scale nuclear interactome profiling

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics...

"cell surface capture" workflow for label-free quantification of the cell surface proteome

&quot;Cell Surface Capture&quot; Workflow for Label-Free Quantification of the Cell Surface Proteome

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized...

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

high-throughput transcriptome analysis for investigating host-pathogen interactions

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical...

in silico identification and characterization of circrnas during host-pathogen interactions

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions

The protocol submitted here explains the complete in silico pipeline needed to predict and functionally characterize circRNAs from RNA-sequencing transcriptome data studying host-pathogen...

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions

quantitative proteomics workflow using multiple reaction monitoring based detection of proteins from human brain tissue

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be...

use of the epiairway model for characterizing long-term host-pathogen interactions

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

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model.  The approach can be used with any microbe that is amenable to long-term...

infection of zebrafish larvae with aspergillus spores for analysis of host-pathogen interactions

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

use of an optical trap for study of host-pathogen interactions for dynamic live cell imaging

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

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope.  The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells.  Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to...

a comparative approach to characterize the landscape of host-pathogen protein-protein interactions

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

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called...

a clinical metaproteomics workflow implemented within galaxy bioinformatics platform to analyze host-microbiome interactions underlying human disease

Author Spotlight: Improving Host-Microbiome Protein Identification and Functional Insights Using the Galaxy Platform

Clinical metaproteomics offers insights into the human microbiome and its contributions to disease. We harnessed the computational power of the Galaxy platform to develop a modular bioinformatics workflow that facilitates complex mass spectrometry-based metaproteomic analysis and characterization of diverse clinical sample types relevant to studies of...

A Clinical Metaproteomics Workflow Implemented within Galaxy Bioinformatics Platform to Analyze Host-Microbiome Interactions Underlying Human Disease

pip-on-a-chip: a label-free study of protein-phosphoinositide interactions

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH...

double labeling immunofluorescence using antibodies from the same species to study host-pathogen interactions

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and...

a quantitative glycomics and proteomics combined purification strategy

A Quantitative Glycomics and Proteomics Combined Purification Strategy

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags...

comprehensive workflow of mass spectrometry-based shotgun proteomics of tissue samples

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a...

quantitative proteomics using reductive dimethylation for stable isotope labeling

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

label-free identification of lymphocyte subtypes using three-dimensional quantitative phase imaging and machine learning

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

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell...

phosphopeptide enrichment coupled with label-free quantitative mass spectrometry to investigate the phosphoproteome in prostate cancer

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based...

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

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

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

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

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

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

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 die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

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

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

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

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 to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

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

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

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

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

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

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

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

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

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

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

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