title: Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies.
abstract: A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspecies-specific biomarker peptides for three Salmonella subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level.
authors.affiliation: Agroscope, Institute for Food Sciences, Waedenswil, Switzerland.
authors.forename: Maria-Theresia
authors.initials: MT
authors.lastname: Gekenidis
authors.affiliation: Agroscope, Institute for Food Sciences, Waedenswil, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Studer
authors.affiliation: Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich, Switzerland.
authors.forename: Simone
authors.initials: S
authors.lastname: Wüthrich
authors.affiliation: Functional Genomics Center Zurich, ETH Zurich and University of Zurich, Zurich, Switzerland.
authors.forename: René
authors.initials: R
authors.lastname: Brunisholz
authors.affiliation: Agroscope, Institute for Food Sciences, Waedenswil, Switzerland david.drissner@agroscope.admin.ch.
authors.forename: David
authors.initials: D
authors.lastname: Drissner
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D015373:Bacterial Typing Techniques / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D003470:Culture Media; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D010641:Phenotype; D010802:Phylogeny; D040901:Proteomics / Q000379:methods; D012475:Salmonella / Q000145:classification* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D010455:Peptides