Synthesis and characterisation of PEG-peptide surfaces for proteolytic enzyme detection

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


Synthesis and characterization of CF-GR for calibration curve.
The peptide CF-GR, which was used to create the calibration curve, was synthesized by SPPS using the Fmoc strategy. The 2-chlorotrityl chloride resin (126 mg, 1.3 mmol/g) was suspended in 0.5 mL of DCM. One equivalent of Fmoc-R(Pbf) (106 mg, 0.168 mmol) was dissolved in 0.5 mL of DCM, and 2.5 equivalents of DIPEA (51 µL, 0.297 mmol) were added to the resin suspension and stirred for 30 min. Next, the resin was washed with MeOH and stirred for 15 min to end cap any remaining reactive 2-chlorotrityl groups. The resin was filtered and washed three times in DCM, twice in DMF and three times in DCM.

Estimation of the Fmoc-R(Pbf) loading capacity:
Ten milligrams of the dry resin was treated with 100 µL of 30% piperidine in DMF for 30 min. Ten millilitres of EtOH was added to the suspension. The average absorbance value (A) measured at 301 nm for three samples were used for the calculation. The resin substitution, S 0 , was calculated using the formula: where ε=5304, -the extinction coefficient of the Fmoc-piperidine adduct measured in EtOH during a separate experiment, V -sample volume, m -mass of the resin, d -width of the cuvette. The calculated resin substitution was equal to S 0 =0.356 mmol/g.
The coupling of Fmoc-G (36 mg, 0.124 mmol) was performed in 0.5 mL of DMF for two hours using 3 equivalents of amino acid, COMU (53 mg, 0.124 mmol) and 6 equivalents of DIPEA (43 µL, 0.248 mmol). The removals of Fmoc were performed using a 20% piperidine solution in DMF (2 times for 10 min). After each coupling and deprotection step, the resin was washed 3 times in DMF, DCM, and again in DMF. (5,6)-Carboxyfluoresceine (CF) (46 mg, 0.124 mmol) was coupled to the N-terminal amine group of the glycine residue using three equivalents of HOBt (19 mg, 0.124 mmol) and DIC (20 µL, 0.124 mmol). Reaction was performed for two hours in DMF. After the addition of CF, the resin was washed (two times for 10 min) with 20% piperidine in DMF. The quantitative yield of each coupling reaction was confirmed by a negative result of the Kaiser test. The peptide was cleaved from the resin by treating with a cleavage mixture (95% TFA, 2.5% TIS and 2.5% H2O) for 4 hours. Excess TFA was removed under an argon flow and then the peptide was precipitated in diethyl ether and isolated by centrifugation. Twenty-one milligrams of the CF-GR peptide with a 98% purity was obtained (determined by RP-HPLC, Fig. S2). The structure of the peptide was confirmed by ESI-MS (

CF-GR calibration curve
The peptide CF-GR was used for the preparation of the fluorescence calibration curve (Fig. S4). The fluorescence measurements were performed in a 50 mM ammonium bicarbonate buffer (pH=7) to maintain the same solvent as that used for the enzymatic reaction.

Modification of silicon surface
The silicon wafers were cleaned with acetone and rinsed several times with deionized water. To oxidize the silicon surface, the wafers were heated to 120 °C and immersed in a piranha solution (1:3, v/v mixture of 30% H 2 O 2 and 95% H 2 SO 4 ). The wafers were stored in the piranha solution for 2 h and subsequently washed with deionized water, acetone, ethanol and then dried.
The freshly prepared hydroxyl-terminated wafers were reacted with 3-aminopropyltriethoxysilane (APTES). The wafers were kept in a 2% EtOH solution of APTES for 2 hours at room temperature. The washed with EtOH wafers were left overnight in an oven at 120 °C. After the reaction, the Si-APTES wafers were rinsed three times with EtOH and dried under an argon flow. After each step of the modification, the wafers were analysed using AFM and CA techniques.

The estimation of amine loading of SI-APTES surface
In order to calculate the amount of amine present on the surface after silinization the reaction of functionalized silicon (Si-APTES) with ninhydrin was performed. The Si-APTES wafers, heated to 120 °C, were covered with a mixture of reagents: 400 µL 5% ninhydrin/n-BuOH, 50 µL 80% phenol/n-BuOH, 50 µL 0.01 M KCN/pyridine (Kaiser test). The reaction was performed for two hours to ensure a quantitative yield of the formed product (Ruhemann's purple). The post-reaction solutions were collected and their absorbance was measured by UV-VIS at 590 nm.

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The number of amino-terminated units per surface area was calculated using the equation: were the extinction coefficient ε=5447 was measured in separated experiment, A -average absorbance, Vsample volume, a -the surface area, d=1 -the width of cuvette.
The calculated amine loading was 3.5 pmol/mm 2 .