Sensor Array Based Determination of Edman Degradated Amino Acids Using Poly(p‐phenyleneethynylene)s

Abstract A cross‐reactive optical sensor array based on poly(p‐phenyleneethynylene)s (PPEs) determines Edman degraded amino acids. We report a sensor array composed of three anionic PPEs P1–P3, and their electrostatic complexes with metal ions (Fe2+, Cu2+, Co2+). We recorded distinct fluorescence intensity response patterns as “fingerprints” of this chemical tongue toward standard phenylthiohydantoin (PTH) amino acids—degradation products of the Edman process. These “fingerprints” were converted into canonical scores by linear discrimination analysis (LDA), which differentiates all of the PTH‐amino acids. This array discriminates PTH‐amino acid residues degraded from an oligopeptide through Edman sequencing. This approach is complementary to chromatography approaches which rely on mass spectrometry; our array offers the advantage of simplicity.


General information
1.1 Materials. Chemicals and solvents were either purchased from the chemical store at the Organisch-Chemisches Institut of the University of Heidelberg or from commercial laboratory suppliers. Standard PTH-amino acids were purchased from TCI or Santa Cruz Biotechnology. Inc.
Metal salts were used as Fe(ClO4)2•xH2O, Cu(ClO4)2•6H2O, and Co(ClO4)2•6H2O. Reagents were used without further purification unless otherwise noted. The synthesis of PPEs P1-P7 were reported previously. [1] 1.2 Method for Fluorescence response pattern. Fluorescence intensity (I0 or I) was recorded on a CLARIOstar (firmware version 1.13) Platereader from BMG Labtech using the corresponding software (software version 5.20 R5). Data were analyzed with CLARIOstar MARS Data Analysis Software (software version 3.10 R5) from BMG Labtech. I0 and I are the fluorescence intensity of the solution in the absence and presence of the PTH-amino acids, respectively. To ensure solubility, stock solutions of all the analyte PTH-amino acid were prepared in DMSO and diluted using water (2 mg/mL). The stock solutions for metal ions (10 mM) and PPEs (P1, P3: 1 mM; P2, 0.4 mM) were prepared in water and diluted using DMSO as needed in the sensing experiments. 150 μL of polymers or the polymer-metal ions solution in DMSO/H2O (1:1) was loaded into a well on a 96well plate (300 μL microplate) first. Subsequently, 150 μL PTH-amino acids solution were added to each well. After incubation for 2 h at room temperature, the fluorescence intensity values were recorded on the microplate reader with an excitation at 410 nm for P1, 430 nm for P2 and P3.
1.3 Linear discriminant analysis (LDA) was carried out using classical linear discriminant analysis in SYSTAT (version 13.0). In LDA, all variables were used in the model (complete mode) and the tolerance was set as 0.001. The fluorescence response patterns were transformed to canonical patterns. The Mahalanobis distances of each individual pattern to the centroid of each group in a multidimensional space were calculated and the assignment of the case was based on the shortest Mahalanobis distance.

Detailed procedure for Edman degradation
Stepwise degradation for oligopeptide was performed by a modification of a previously described method. [2] 3.

Preparation of the PTC-peptide
To a glass centrifuge tube, 28.9 mg of Met-Ala-Ser was added and fully dissolved in 2 mL pyridine-water (1:1, v/v, pH ca. 8.8). Under nitrogen atmosphere, 200 μL phenyl isothiocyanate (PITC) was added to the solution of peptide. The reaction mixture was vigorously stirred (1000 rpm) for 30 minutes in a 50 o C water bath. After the completion of the reaction, the mixture was cooled down to 0 o C. The PTC-derivative was thoroughly washed and extracted using benzene (5-8 times) and the phases were separated by centrifuge for 3 min at 3500 rpm. The excess PITC and side products in upper phase was removed and the water phase was concentrated under a stream of nitrogen, and then dried in vacuum.

Cleavage of the PTC-peptide
Under nitrogen atmosphere, the dried PTC-peptide was treated with anhydrous TFA (300 μL).
The cleavage reaction was carried out in a water bath (50 o C, 10 min.). The TFA was removed using a flow of nitrogen and then dried under vacuum. The thiazoline derivative (ATZ-amino acid) was extracted from the remaining peptide with ethyl acetate/H2O (2:1, v/v, 3 mL). The remaining peptide in water phase (1 mL) was coupled immediately for next round degradation (see 3.1) or kept in the freezer. The ATZ-amino acid in the ethyl acetate phase was flushed with N2 and dried under vacuum.

Transformation of the ATZ-amino acid
Under nitrogen atmosphere, the dried ATZ-amino acid was dispersed in 300 μL 1N HCl and the conversion reaction was carried out in a water bath (80 o C, 10 min.). After the completion of the reaction, the mixture was cooled down to 0 o C. The mixture was extracted 3 times with ethyl acetate and the organic phase containing PTH-amino acid was collected after centrifugation. The ethyl acetate layer was washed with saturated sodium bicarbonate solution followed by washing with