title: Comparison of five methods for direct extraction of surface proteins from Listeria monocytogenes for proteomic analysis by orbitrap mass spectrometry.
abstract: Extracts of surface proteins, with minimal artifacts from contaminating cytosolic components, are highly desirable for investigating surface factors involved in the attachment and formation of biofilms by bacteria that are problematic in commercial food processing facilities. In this study, we compared the protein profiles of the food pathogen, Listeria monocytogenes, recovered after applying different surface protein extraction methods compiled from the literature: trypsin-enzymatic shaving with BICAM/sucrose or Tris/sucrose buffers (Tryp B+S, Tryp T+S), Tris-buffered urea (UB), lithium chloride (LiCl) and Tris-buffered urea applied with hypotonic-stressed cells (UB-Ghost), and subjected them to liquid chromatography tandem mass spectrometry and protein identification. The data indicate that the UB-Ghost extraction method provides a cleaner extract of surface proteins including the predicted (this study and the literature) or validated members (literature) from L. monocytogenes. This was determined by an accumulative lower unique peptide number exhibited by mass spectrometry for total cytoplasmic proteins among different surface extracts, with a majority of proteins demonstrating hydrophilic properties. The extracted proteins were from different functional categories and have associations with the cell surface, intermediary metabolism, information pathways, or functionally unknown proteins as suggested by in silico analyses performed by other groups (Leger and ListiList). The utilization of an optimized method for surface protein extraction should greatly facilitate identification by LC-MS/MS that could be useful to anyone working on molecular proteomics of bacterial surfaces.
authors.affiliation: Dept. of Animal Sci., Oklahoma State University, 109 FAPC Bldg, Monroe St., Stillwater, OK 74078-6055, United States; The Robert M. Kerr Food & Agricultural Products Ctr., Oklahoma State University, 307 FAPC Bldg, Monroe St., Stillwater, OK 74078-6055, United States. Electronic address: htiong@ostatemail.okstate.edu.
authors.forename: Hung King
authors.initials: HK
authors.lastname: Tiong
authors.affiliation: Dept. Biochem. Molec. Biol., Oklahoma State University, Stillwater, OK 74078, United States. Electronic address: steven.hartson@okstate.edu.
authors.forename: Steven
authors.initials: S
authors.lastname: Hartson
authors.affiliation: Dept. of Animal Sci., Oklahoma State University, 109 FAPC Bldg, Monroe St., Stillwater, OK 74078-6055, United States; The Robert M. Kerr Food & Agricultural Products Ctr., Oklahoma State University, 307 FAPC Bldg, Monroe St., Stillwater, OK 74078-6055, United States. Electronic address: peter.muriana@okstate.edu.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Muriana
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D008089:Listeria monocytogenes / Q000737:chemistry*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D014357:Trypsin
keywords: Adherence; Extraction; Listeria monocytogenes; Mass spectrometry; Proteomics; Surface proteins