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. 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>

The authors declare no conflicts of interest.

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 Views.  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 ...