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.FigureMonitoring of trypsin hydrolysis on PEG-peptide surfaceElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-013-7082-z) contains supplementary material, which is available to authorized users.

An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

“…TOF-SIMS has been already used in the steric analysis of proteins,[ 16 ] lipid–lipid and lipid–protein interactions,[ 186 , 187 ] biomarkers [ 188 ] as well as enzyme immobilization. [ 189 ] For the purpose of characterization of organic samples, the less damaging TOF-SIMS static measurement mode is used as the samples are sputtered softly with ions with low current densities that leave the sample structure intact. The dynamic mode is preferred when extreme high sensitivity analysis is required in which ions of high current densities are used to bombard the sample surface, which in tern leads to damaging the surface.…”

Co-Eradication of Breast Cancer Cells and Cancer Stem Cells by Cross-Linked Multilamellar Liposomes Enhances Tumor Treatment

“…A surface modification was made on each of the cMLV formulations by adding thiol-termineated polyethylene glycol (PEG) chains, as previously described 22,23 . The conjugation of polyethylene glycol (PEGylation) to nanoparticles is an effective way to enhance drug delivery to the site of interest by further enhancing the plasma half-life and serum stability of the vesicles [42][43][44] . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 To characterize the cMLVs, the particle size was measured by dynamic light scattering (DLS).…”

A single amino acid Gly-tag enables metal-free protein purification

“…The PEG linker regulates the surface availability of the reagent upon its immobilization. 22 On the other hand, the amine functionality provides the nucleophilic handle to conjugate it with the electrophilic NHS ester functionalized resin (4). Next, we quantied the extent of immobilization of the Gly-capture reagent on the Sepharose resin by UV analysis (Fig.…”