title: 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.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Kristin E
authors.identifier: http://orcid.org/0000-0002-2722-4149
authors.initials: KE
authors.lastname: Burnum-Johnson
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Kyle
authors.affiliation: Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Amie J
authors.initials: AJ
authors.lastname: Eisfeld
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Cameron P
authors.initials: CP
authors.lastname: Casey
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Kelly G
authors.initials: KG
authors.lastname: Stratton
authors.affiliation: Department of Microbial Infection and Immunity, Ohio State University, Columbus, OH, USA.
authors.forename: Juan F
authors.initials: JF
authors.lastname: Gonzalez
authors.affiliation: Department of Microbial Infection and Immunity, Ohio State University, Columbus, OH, USA.
authors.forename: Fabien
authors.initials: F
authors.lastname: Habyarimana
authors.affiliation: School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Negretti
authors.affiliation: Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: Amy C
authors.initials: AC
authors.lastname: Sims
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Sadhana
authors.initials: S
authors.lastname: Chauhan
authors.affiliation: Departments of Medicine, Molecular Microbiology, Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, USA.
authors.forename: Larissa B
authors.initials: LB
authors.lastname: Thackray
authors.affiliation: Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Halfmann
authors.affiliation: Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Kevin B
authors.initials: KB
authors.lastname: Walters
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Young-Mo
authors.initials: YM
authors.lastname: Kim
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Erika M
authors.initials: EM
authors.lastname: Zink
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Carrie D
authors.initials: CD
authors.lastname: Nicora
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Karl K
authors.initials: KK
authors.lastname: Weitz
authors.affiliation: Computational and Statistical Analytics Division, Pacific Northwest National Laboratory, Richland, WA, USA.
authors.forename: Bobbie-Jo M
authors.initials: BM
authors.lastname: Webb-Robertson
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Ernesto S
authors.initials: ES
authors.lastname: Nakayasu
authors.affiliation: Department of Microbial Infection and Immunity, Ohio State University, Columbus, OH, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Ahmer
authors.affiliation: School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Konkel
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Motin
authors.affiliation: Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: Ralph S
authors.initials: RS
authors.lastname: Baric
authors.affiliation: Departments of Medicine, Molecular Microbiology, Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Diamond
authors.affiliation: Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kawaoka
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Katrina M
authors.initials: KM
authors.lastname: Waters
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA. thomas.metz@pnnl.gov.
authors.forename: Thomas O
authors.identifier: http://orcid.org/0000-0001-6049-3968
authors.initials: TO
authors.lastname: Metz
mesh_terms: D001419:Bacteria / Q000302:isolation & purification*; D002460:Cell Line; D004847:Epithelial Cells; D006801:Humans; D008055:Lipids / Q000032:analysis*; D013058:Mass Spectrometry; D055432:Metabolomics; D011506:Proteins; D040901:Proteomics; D038301:Virus Inactivation; D014780:Viruses / Q000302:isolation & purification*
chemical_list: D008055:Lipids; D011506:Proteins