Ion-Transfer Voltammetric Behavior of Protein Digests at Liquid|Liquid Interfaces

Abstract

The development of new methods for the detection of proteins and peptides is of widespread importance. In this work, the electrochemical behavior of peptide mixtures resulting from proteolytic digestion of proteins was investigated at the polarized liquid|liquid interface (or the interface between two immiscible electrolyte solutions, ITIES). The influence of pepsin digestion on three proteins (hemoglobin, lysozyme, and cytochrome c) was studied, and it was revealed that resulting cyclic voltammograms of the three protein digests were different due to the unique peptide mixtures for a given protein. Differential pulse stripping voltammetry of protein digests enabled the detection of digested proteins at concentrations ranging between 0.55 and 4.22 microM. A limit of detection of 0.55 microM of the initial concentration of protein was achieved, demonstrating the analytical possibilities of such an electrochemical method. These results show that ion-transfer voltammetry offers the opportunity to study and develop label-free detection of peptides resulting from enzymatic digestions of proteins and may thus have a role in development of new proteomic technologies.

Voltammetry of proteins at liquid–liquid interfaces

“…60 Hartvig et al 52 employed an online mass spectrometry method to detect protein-anion Herzog et al investigated this scenario, by denaturing proteins with urea 61 and by enzymatic digestion. 62 Indeed, it was found that the signals for these denatured or digested proteins were different from those of the native proteins. However, it must be borne in mind that when examining the electrochemistry of a protein in an acidic aqueous phase in contact with an organic phase, there is considerable scope for at least partial denaturation in the acidic phase and in contact with the interface, irrespective of any intentional denaturation using regular denaturants such as urea.…”

Liquid/Liquid Interfaces, Electrochemistry atUpdate based on the original article by Frédéric Reymond, Hubert H. Girault,Encyclopedia of Analytical Chemistry, © 2000, John Wiley & Sons, Ltd

“…Electrochemistry at ITIES has also been used to investigate chemical denaturation, (149) enzymatic proteolysis of proteins, (150) and protein unfolding. (151) Complexation of DNA with acridine-functionalized calixarene has also been investigated at ITIES.…”

Haemoglobin unfolding studies at the liquid–liquid interface

“…These interactions between lipophilic anions and proteins adsorbed at the interface were confirmed by biphasic electrospray ionization mass spectrometry [17], where adsorbed lysozyme molecules form complexes with tetrakis(4-chlorophenyl)borate (TPBCl -) of stoichiometry 1:1, 1:2 and 1:3 (lysozyme:TPBCl -). Recent studies have demonstrated that the electrochemical response of proteins at the ITIES is greatly influenced by disruptions of their structure, whether achieved by the presence of a chemical denaturant [18] or by proteolysis [19]. In the presence of urea, the electrochemical behaviour of Hb was greatly altered leading to a lower peak current and charge [18].…”