title: Synthesis and characterisation of PEG-peptide surfaces for proteolytic enzyme detection.
abstract: Peptide surfaces were obtained by the covalent immobilisation of fluorescently labelled pentapeptides carboxyfluorescein-glycine-arginine-methionine-leucine-glycine, either directly or through a poly(ethylene glycol) (PEG) linker on modified silicon wafers. Each step during the preparation of the peptide surfaces was confirmed by several surface characterisation techniques. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy were used to determine the surface composition, the wafers philicity was measured by contact angle and atomic force microscopy was used to investigate the surface morphology. Exposure of the peptide surfaces to trypsin resulted in the release of a fluorescently labelled peptide product, which allowed the kinetics of the enzymatic reaction to be followed with the aid of fluorescence spectroscopy. The electrospray ionisation mass spectrometry analysis of the post-digestion solution confirmed that the pentapeptides attached to the solid support undergo specific trypsin hydrolysis at the C-terminus of the arginine residues. Detailed surface analyses before and after the enzyme action was performed using ToF-SIMS. Because of the limited accessibility of the short peptide directly attached to the surface, a quantitative yield of enzymatic hydrolysis was observed only in case when the peptide was bound through the PEG linker. The insertion of the PEG linker increased the number of immobilised peptides and the rate of enzymatic digestion which consequently improved the quality of the enzyme assays. The described approach may be used for different peptide sequences designed for other proteases.
authors.affiliation: Centre of Polymer and Carbon Materials, Polish Academy of Sciences, M. Curie-Sklodowskiej 34, Zabrze, 41-819, Poland.
authors.forename: Roza
authors.initials: R
authors.lastname: Trzcinska
authors.forename: Piotr
authors.initials: P
authors.lastname: Suder
authors.forename: Anna
authors.initials: A
authors.lastname: Bodzon-Kulakowska
authors.forename: Magdalena
authors.initials: M
authors.lastname: Skalska
authors.forename: Andrzej
authors.initials: A
authors.lastname: Marcinkowski
authors.forename: Jerzy
authors.initials: J
authors.lastname: Kubacki
authors.forename: Roman
authors.initials: R
authors.lastname: Pedrys
authors.forename: Jerzy
authors.initials: J
authors.lastname: Silberring
authors.forename: Andrzej
authors.initials: A
authors.lastname: Dworak
authors.forename: Barbara
authors.initials: B
authors.lastname: Trzebicka
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D010447:Peptide Hydrolases / Q000032:analysis*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*
chemical_list: D010455:Peptides; D011092:Polyethylene Glycols; D010447:Peptide Hydrolases