Software routine allowing identification of extracellular ... · As demonstrated by the experiments...

KIT – University of the State of Baden-Wuerttemberg andNational Research Center of the Helmholtz Association

AIM OF THE STUDYThe 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.

Siegfried Hohmann , Anke Neidig, Boris Kuehl, Frank Kirschhoefer, Joe rg Overhage , Gerald Brenner-Weiss *

Software routine allowing identification of Software routine allowing identification of extracellularextracellular biofilmbiofilm proteins proteins involved in cell adhesion based on data from QCMinvolved in cell adhesion based on data from QCM --D/MALDI experimentsD/MALDI experiments

Department of microbiology of natural and technical surfaces , Institute of Functional Interfaces (IFG)

CONCLUSIONSAs 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.

CONTACTe-mail: gerald.brenner-weiss@kit.edu

RESULTS AND DISCUSSIONWe 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.

INTRODUCTIONThe 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.

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IWA conference “The perfect slime” 2014

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MATERIALS AND METHODSSupernatant of one and three days cultured P. aeruginosa ( ) was adsorbed onto quarz-crystal-microbalance (QCM-D) chips ( ) and the adsorbed mass was monitored ( ).

The adsorbed layer was digested by trypsin ( ) and concentrated using chromatographic material ( ) contained in a pipette tip (ZipTip®).

After concentration the sample was spotted onto a MALDI target plate and mass spectra of the contained peptides ( ) were generated.

Using algorithms implemented in PHP the peak lists generated from the mass spectra were analysed and matched against theoretical trypticaldigests of all proteins encoded in the genome of PAO1 generated by scripts and stored in an xml based data base system ( ).

From the matched proteins identified in three independent experiments of different bacterial cultures ( ), selected peaks were further analysed using MALDI-ToF/MSMS ( ).

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The obtained MS-MS spectra were matched against theoretical fragmentations of the peptide sequences ( ) to ensure the identity of the protein.A scoring system ( ) was used to sort the found proteins by their probability of identification.

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