Software routine allowing identification of extracellular biofilm proteins involved in cell adhesion based on data from QCM-D/MALDI experiments Siegfried Hohmann, Anke Neidig, Boris Kuehl, Frank Kirschhoefer, Joerg Overhage, Gerald Brenner-Weiss* Department of microbiology of natural and technical surfaces, Institute of Functional Interfaces (IFG) INTRODUCTION The first step in the settelment of planctonic bacterial from a liquid medium onto a solid surface is the adsorption of substances contained in the medium onto the surface. The layer of adsorbed substances, e.g. proteins, polysaccharides, etc. is called conditioning film. mass mass A B C time intensity mass mass mass mass intensity intensity mass 3x intensity time 3x mass intensity mass 3x 3x intensity time intensity intensity intensity mass 3x 3x AIM OF THE STUDY The aim of the study was to determine which kind of proteins from cell free Pseudomonas aeruginosa PAO1 supernatant adsorb onto our model surface TiO2, which is often used as material for transplants. mass mass F D E MATERIALS AND METHODS Supernatant of one and three days cultured P. aeruginosa (A) was adsorbed onto quarz-crystal-microbalance (QCM-D) chips ( B) and the adsorbed mass was monitored (C). The adsorbed layer was digested by trypsin ( D) and concentrated using chromatographic material (E) contained in a pipette tip (ZipTip®). After concentration the sample was spotted onto a MALDI target plate and mass spectra of the contained peptides ( F) were generated. Using algorithms implemented in PHP the peak lists generated from the mass spectra were analysed and matched against theoretical tryptical digests of all proteins encoded in the genome of PAO1 generated by scripts and stored in an xml based data base system (G). From the matched proteins identified in three independent experiments of different bacterial cultures (H), selected peaks were further analysed using MALDI-ToF/MSMS ( I ). 840.4769 845.4564 848.4493 856.5310 861.0806 … PA0027 PA0040 PA0041 PA0077 PA0090 … 815.5227 825.1055 845.4537 849.4158 856.5280 … PA0004 PA0027 PA0040 PA0041 PA0044 … 845.4611 848.4566 849.4420 856.5380 861.0865 … G H The obtained MS-MS spectra were matched against theoretical fragmentations of the peptide sequences ( J ) to ensure the identity of the protein. A scoring system ( K ) was used to sort the found proteins by their probability of identification. RESULTS AND DISCUSSION We were able to identify several intracellular as well as extracellular proteins including adhesion proteins to be involved in conditioning film formation in all three supernatants of P. aeruginosa PAO1 by MALDI-ToF/MS and to ensure their correct identification by MALDI-ToF/MSMS. CONCLUSIONS As demonstrated by the experiments the coupling of QCM-D with high resolution mass spectrometry (MALDI-ToF/MS) is a powerful tool for analyzing conditioning films of supernatant of P. aeruginosa. PA0040 PA0041 PA0101 PA0413 PA0690 … J I PA0007 PA0040 PA0041 PA0074 PA0101 … KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association K CONTACT e-mail: [email protected] IWA conference “The perfect slime” 2014