title: Small protein biomarkers of culture in Bacillus spores detected using capillary liquid chromatography coupled with matrix assisted laser desorption/ionization mass spectrometry.
abstract: Capillary liquid chromatography (cLC) coupled with matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) was used to compare small proteins and peptides extracted from Bacillus subtilis spores grown on four different media. A single, efficient protein separation, compatible with MALDI-MS analysis, was employed to reduce competitive ionization between proteins, and thus interrogate more proteins than possible using direct MALDI-MS. The MALDI-MS data files for each fraction are assembled as two-dimensional data sets of retention time and mass information. This method of visualizing small protein data required careful attention to background correction as well as mass and retention time variability. The resulting data sets were used to create comparative displays of differences in protein profiles between different spore preparations. Protein differences were found between two different solid media in both phase bright and phase dark spore phenotype. The protein differences between two different liquid media were also examined. As an extension of this method, we have demonstrated that candidate protein biomarkers can be trypsin digested to provide identifying peptide fragment information following the cLC-MALDI experiment. We have demonstrated this method on two markers and utilized acid breakdown information to identify one additional marker for this organism. The resulting method can be used to identify discriminating proteins as potential biomarkers of growth media, which might ultimately be used for source attribution.
authors.affiliation: Chemical and Biological Sciences, Pacific Northwest National Laboratory, Richland, WA 99354, United States.
authors.forename: David
authors.initials: D
authors.lastname: Wunschel
authors.forename: Jon
authors.initials: J
authors.lastname: Wahl
authors.forename: Alan
authors.initials: A
authors.lastname: Willse
authors.forename: Nancy
authors.initials: N
authors.lastname: Valentine
authors.forename: Karen
authors.initials: K
authors.lastname: Wahl
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry*; D001426:Bacterial Proteins / Q000032:analysis*; D015415:Biomarkers / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013171:Spores, Bacterial / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D015415:Biomarkers