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

Summary

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.

Abstract

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.

Introduction

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 meningoencephalitis^2,3. The global increase in immunocompromised status has demanded a parallel increase in the use ....

Protocol

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 (Penicillin-Streptomycin) mixture to DMEM (Dulbecco's Modified Eagle Medium) and 20% DMSO (dimethyl sulfoxide).

1. Culturing of

Results

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 .......

Discussion

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 .......

Materials

List of materials used in this article

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-Art™ 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	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