MPLEx: a method for simultaneous pathogen inactivation and extraction of samples for multi-omics profiling

Abstract

The continued emergence and spread of infectious agents is of great concern, and systems biology approaches to infectious disease research can advance our understanding of host-pathogen relationships and facilitate the development of new therapies and vaccines. Molecular characterization of infectious samples outside of appropriate biosafety containment can take place only subsequent to pathogen inactivation. Herein, we describe a modified Folch extraction using chloroform/methanol that facilitates the molecular characterization of infectious samples by enabling simultaneous pathogen inactivation and extraction of proteins, metabolites, and lipids for subsequent mass spectrometry-based multi-omics measurements. This single-sample metabolite, protein and lipid extraction (MPLEx) method resulted in complete inactivation of clinically important bacterial and viral pathogens with exposed lipid membranes, including Yersinia pestis, Salmonella Typhimurium, and Campylobacter jejuni in pure culture, and Yersinia pestis, Campylobacter jejuni, and West Nile, MERS-CoV, Ebola, and influenza H7N9 viruses in infection studies. In addition, >99% inactivation, which increased with solvent exposure time, was also observed for pathogens without exposed lipid membranes including community-associated methicillin-resistant Staphylococcus aureus, Clostridium difficile spores and vegetative cells, and adenovirus type 5. The overall pipeline of inactivation and subsequent proteomic, metabolomic, and lipidomic analyses was evaluated using a human epithelial lung cell line infected with wild-type and mutant influenza H7N9 viruses, thereby demonstrating that MPLEx yields biomaterial of sufficient quality for subsequent multi-omics analyses. Based on these experimental results, we believe that MPLEx will facilitate systems biology studies of infectious samples by enabling simultaneous pathogen inactivation and multi-omics measurements from a single specimen with high success for pathogens with exposed lipid membranes.

Metabolite, Protein, and Lipid Extraction (MPLEx): A Method that Simultaneously Inactivates Middle East Respiratory Syndrome Coronavirus and Allows Analysis of Multiple Host Cell Components Following Infection

“…(e) Retention time: verify that it is within allowable tolerances for LC system. 6. Select the correct identifications as going through step 5.…”

“…For example, samples from MERS-CoV infected patients are virtually unavailable for analysis, and animal models that recapitulate disease phenotypes seen in humans have only recently been generated, both of which have drastically slowed our progress toward understanding MERS-CoV pathogenesis in the host [3]. Systems biology studies offer a way to capture big picture snapshots of individual cellular components (proteins, lipids, metabolites) that are modulated over the course of infection to develop a better understanding of pathogen-host interactions [4][5][6][7][8][9][10][11][12][13].…”

“…While it's clear that metabolites, proteins, and lipids play an important role in fully characterizing the MERS-CoV infection, the proteomic, metabolomic, and lipidomic sample manipulation of MERS-CoV outside of appropriate biosafety level (BSL) containment laboratories can take place only subsequent to pathogen inactivation. Here, we describe the MPLEx (metabolite, protein, and lipid extraction) protocol for the extraction of protein, metabolites, and lipids from a single sample that simultaneously inactivates the MERS-CoV virus [5,6,26]. Each analyte can then be analyzed by the respective mass spectrometry (MS)-based omics pipeline.…”

“…To illustrate the effectiveness of this protocol, Nakayasu et al performed an integrative multi-omics study using a human lung epithelial cell line infected with MERS-CoV, which showed the impact of the viral infection on the host glycolytic pathway, different host metabolic pathways, and also global changes in lipid profiles induced by infection [5]. To illustrate the effectiveness of MPLEx on pathogen inactivation, Burnum-Johnson et al showed complete inactivation of both bacterial and viral pathogens with exposed lipid envelopes, including MERS-CoV [6].…”

“…Repurposed from Ref. [6] with permission from The Royal Society of Chemistry 11. Add 200 μL 100% methanol to the protein "disc" and shake vigorously to mix.…”