Disposable Carbon Screen-Printed Electrodes for On-Chip Protein Digestion: A Proteomic Approach Coupled to MALDI-TOF MS

Proteomics aims to achieve the large-scale characterization and quantification of proteins within complex biological systems. In mass spectrometry (MS)-based proteomics, the bottom-up approach, where proteins are proteolyzed into peptides, remains predominant due to the enhanced detectability of peptides. Conventional proteolysis relies on enzymatic or chemical reactions, whereas electrochemical proteolysis represents a promising, low-cost, eco-friendly, and rapid alternative. However, its implementation remains limited due to extensive protein adsorption on electrode surfaces, which is primarily governed by hydrophobic interactions that hinder electrochemical activity. In this work, we developed an electrochemical proteolysis platform employing carbon screen-printed electrodes (SPEs) directly coupled to MS detection. The main challenges addressed were (i) reducing protein adsorption on carbon surfaces to enhance proteolytic efficiency and (ii) enabling direct coupling of the electrolyzed sample to the MS without purification. To decrease surface hydrophobicity, the electrodes underwent electrochemical and plasma treatments, characterized by contact angle measurements (CAM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Electrochemically treated SPEs exhibited superior stability (30 days), an enhanced heterogeneous rate constant (<i>k</i>⁰ = (2.01 ± 0.15) × 10^-4 cm²/s), and a larger electroactivity area (<i>A</i>_e = 0.47 ± 0.01 cm²). Reduced BSA adsorption confirmed the improved surface properties. Using this platform, insulin was successfully digested in a one-pot electrochemical process (-1.2 V, 30 min; +1.1 V, 20 min) and directly analyzed by MALDI-TOF MS. These findings demonstrate for the first time the feasibility of carbon SPE-based electrochemical proteolysis, enabling miniaturized, inexpensive, and chemical-free protein digestion compatible with MS-based bottom-up proteomics.