Application of Capillary and Microchip Electrophoresis to Investigate Oxidative Modifications of Proteins and Oxidative Stress

Palestrante: Profa. Dra. Susan M. Lunte – University of Kansas, Lawrence – USA.

Abstract: Oxidative stress is involved in many diseases including Alzheimer’s Disease, autoimmune disease and cancer. Reactive nitrogen and oxygen species (RNOS) are generated during oxidative stress and can react with proteins and other macromolecules in vivo and cause cell damage. In this presentation, the use of capillary (CE) and microchip electrophoresis (ME) for the determination of RNOS and their reaction products with amino acids and proteins is presented. Using this separation-based approach, it is possible to monitor multiple species simultaneously and resolve both endogenous and exogenous interferences. Both these techniques require small volumes, exhibit fast analysis times and yield high separation efficiencies. This makes it possible to monitor transient RNOS directly, as well as investigate the production of RNOS in single cells. A variety of detection methods can be employed with CE and ME including UV, fluorescence and electrochemical detection. ME with LIF detection has been evaluated for the indirect determination of RNOS in microglia lysates following reactions with RNOS specific dyes [1]. The electrophoretic separation makes it possible to monitor several RNOS in a single run, as well as to isolate the reaction product from potential interferences.CE and ME with electrochemical detection (EC) is particularly well suited to studies of oxidative stress since redox reactions are involved in their production. Both peroxynitrite and nitric oxide have been detected directly using ME-EC [2-3]. In these studies, the use of ME-EC for the separation and selective detection of RNOS modified amino acids and peptides was evaluated. Oxidative ME-EC was employed for the separation and selective detection of the products of tyrosine and phenylalanine with the hydroxy radical using ligand exchange MEKC [4]. Since there are very few reducible analytes in biological samples, reductive ME-EC was also investigated for the selective detection of nitrotyrosine containing species in the presence of a large amount of tyrosine and potential interferences. Lastly, a method combining ME with bipolar electrochemistry and chemiluminescence detection was developed as an approach to improve the limits of detection for reductive ME-EC. Bibliographic references: 1. Caruso, G., Benatti, C., Musso, N., Fresta, C. G., Fidilio, A., Spampinato, G., Brunello, N., Bucolo, C., Drago, F., Lunte, S. M., Peterson, B. R., Tascedda, F., and Caraci, F., Biomedicines, (2021) 9(5) 477. 2. Gunasekara, D.B., Siegel, J., Caruso, G., Hulvey, M.K., and Lunte, S.M Analyst, (2014) 139 (13) 3265-3273; 3. Schilly, K.M., Gunawardhana, S.M., Wijesinghe, M.B. and Lunte, S.M., Analytical and Bioanalytical Chemistry, (2020) 412 (24) 6101-6119; 4. Weerasekara D.B. and Lunte, S.M., Electroanalysis, (2022) 34 (12) 1913-1927

Prof. Responsável: José Alberto Fracassi da Silva