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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’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 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 ....
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.......
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 .......
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 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 – The Jarislowsky Fellowship Discovery Award, New Frontiers Research Fund – Exploration (NFRFE-2019-00425), and the Canadian Foundation for Innovation (JELF 38798) for J.G.M., as well as NSERC Canada Graduate Scholarship – Masters and Ontario Graduate Scholarship for B.B., and Queen Elizabeth II Graduate Schol....
Name | Company | Catalog Number | Comments |
100 mM Tris-HCl, pH 8.5 | Fisher Scientific | BP152-1 | Maintain at 4°C |
60 x 15 mm Dish, Nunclon Delta | ThermoFisher Scientific | 174888 | |
6-well cell culture plate | ThermoFisher Scientific | 140675 | |
Acetonitrile, MS grade | Pierce | TS-51101 | |
Acetic Acid | Sigma Aldrich | 1099510001 | |
Acetone | Sigma Aldrich | 34850-1L | |
Ammonium bicarbonate (ABC) | ThermoFisher Scientific | A643-500 | Prepare a stock 50 mM ABC solution, stable at room temperature for up to one month. |
Bel-Artâ„¢ HiFlow Vacuum Aspirator Collection System | Fisher Scientific | 13-717-300 | Not essential, serological pipettes can be used to remove media. |
C18 resin | 3M Empore | 3M2215 | |
Cell Scrapers | VWR | 10062-906 | Not essential, other methods to release macrophage cells can be used. |
Centrifugal vaccuum concentrator | Eppendorf | 07-748-15 | |
Complete Filtration Unit | VWR | 10040-436 | |
Conical falcon tubes (15 mL) | Fisher Scientific | 05-539-12 | |
Countess II Automated Cell Counter | ThermoFisher Scientific | AMQAX1000 | Not essential, haemocytometer can be used as an alternative. |
CytoTox 96 Non-Radioactive Cytotoxicity Assay | Promega | G1780 | |
Dithiothreitol (DTT) | ThermoFisher Scientific | R0861 | Prepare bulk stock solution of 1 M DTT, flash frozen and stored at -20 °C until use. Discard after each use (do not freeze-thaw repeatedly). |
DMEM, high glucose, GlutaMAX Supplement | ThermoFisher Scientific | 10566016 | |
Fetal Bovine Serum (FBS) | ThermoFisher Scientific | 12483020 | Heat inactivate by incubating at 60°C for 30 minutes. Prepare 50 ml aliquots and flash freeze. Thaw prior to media preparation |
Haemocytometer | VWR | 15170-208 | |
HEPES | Sigma Aldrich | H3375 | Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20°C until use. Discard after each use (do not freeze-thaw repeatedly). |
High-performance liquid chromatography system | ThermoFisher Scientific | LC140 | Gradient length is based on sample complexity, recommended 120 min gradient for infectome samples. |
High-resolution mass spectrometer | ThermoFisher Scientific | 726042 | |
Iodoacetamide (IAA) | Sigma Aldrich | I6125 | Prepare 0.55 M bulk stock solution, flash frozen, store at -20°C until use. Discard after each use (do not freeze-thaw repeatedly). |
L-glutamine | ThermoFisher Scientific | 25030081 | Can be aliquot and frozen for storage. Thaw prior to media preparation. |
LoBind Microcentrifuge tubes | Eppendorf | 13-698-794 | |
MaxQuant | https://maxquant.org/ | MaxQuant is a public platform that offers tutorials, such as the MaxQuant Summer School, outlining the computational analysis steps of large MS data sets | |
Microcentrifuge | Eppendorf | 13864457 | |
Penicillin : Streptomycin 10k/10k | VWR | CA12001-692 | Can be aliquot and frozen for storage. Thaw prior to media preparation. |
Peptide separation columns | ThermoFisher Scientific | ES803 | |
Perseus Software | http://maxquant.net/perseus/ | ||
Phosphate Buffered Saline | VWR | CA12001-676 | Puchase not required. PBS can also be prepared but sterile filteration must be performed before use. |
Pierce BCA Protein Assay | ThermoFisher Scientific | Â 23225 | |
Pipette, Disposable Serological (10 mL) | Fisher Scientific | 13-678-11E | |
Pipette, Disposable Serological (25 mL) Basix | Fisher Scientific | 14955235 | |
Probe sonciator | ThermoFisher Scientific | 100-132-894 | |
Protease inhibitor cocktail tablet | Roche | 4693159001 | |
Sodium dodecyl sulfate | ThermoFisher Scientific | 28364 | 20% (w/v) |
Spectrophotometer (Nanodrop) | ThermoFisher Scientific | ND-2000 | |
STAGE tipping centrifuge | Sonation | STC-V2 | |
Thermal Shaker | VWR | NO89232-908 | |
Trifluoroacetic acid | ThermoFisher Scientific | 85183 | |
Trypsin/Lys-C protease mix, MS grade | Pierce | A40007 | Maintain at -20 °C. |
Ultrasonic bath | Bransonic | A89375-450 | Stored in cold room (4C) |
Urea | Sigma Aldrich | U1250-1KG | Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20 °C until use. Discard after each use (do not freeze-thaw repeatedly). |
Yeast-extract peptone dextrose broth | BD Difco | BM20 |
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