Electrochemical Quantification of Enkephalin Peptides Using Fast-Scan Cyclic Voltammetry

Endogenous opioid neuropeptides serve as important chemical signaling molecules in both the central and peripheral nervous systems, but there are few analytical tools for directly monitoring these molecules in situ. The opioid peptides share the amino acid motif, Tyr-Gly-Gly-Phe-, at the N-terminus. Met-enkephalin is a small opioid peptide comprised of only five amino acids with methionine (Met) incorporated at the C-terminus. Tyrosine (Tyr) and Met are electroactive, and their distinct electrochemical signatures can be utilized for quantitative molecular monitoring. This work encompasses a thorough voltammetric characterization of Tyr and Met redox chemistry as individual amino acids and when incorporated into small peptide fragments containing the shared Tyr-Gly-Gly-Phe- motif. NMR spectroscopy was used to determine the structure and conformation at near-physiological conditions. Voltammetric data demonstrate how the peak oxidation potential and the rate of electron transfer are dependent on the local chemical environment. Both the proximity of the electroactive residue to the C- or N-terminus and the hydrophobicity of the additional nonelectroactive amino acids profoundly affect sensitivity. Finally, the work uses the electrochemical signal for individual amino acids in a "training set", with a combination of principal component analysis and least-squares regression to accurately predict the voltammetric signal for short peptides comprising different combinations of those amino acids. Overall, this study demonstrates how fast-scan cyclic voltammetry can be utilized to discriminate between peptides with small differences in the chemical structure, thus establishing a framework for reliable quantification of small peptides in a complex signal, broadly speaking.