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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Mass spectrometry (MS)-based proteomics commonly uses the bottom-up approach, relying on peptide generation via proteolysis.
• Conventional proteolysis methods (enzymatic, chemical) face limitations, driving interest in alternatives like electrochemical proteolysis.
• Protein adsorption onto electrode surfaces hinders electrochemical proteolysis efficiency, primarily due to hydrophobic interactions.

Purpose of the Study:

• To develop a novel electrochemical proteolysis platform using carbon screen-printed electrodes (SPEs) directly coupled to MS detection.
• To overcome challenges of protein adsorption on carbon surfaces for improved proteolytic efficiency.
• To enable direct MS analysis of electrolyzed samples without intermediate purification steps.

Main Methods:

• Carbon SPEs were treated electrochemically and via plasma to reduce surface hydrophobicity.
• Surface modifications were characterized using contact angle measurements (CAM), SEM, XPS, CV, and EIS.
• Insulin digestion was performed in a one-pot electrochemical process and analyzed directly by MALDI-TOF MS.

Main Results:

• Electrochemical treatment significantly improved SPE stability (30 days), heterogeneous rate constant (k0 = (2.01 ± 0.15) × 10⁻⁴ cm²/s), and electroactivity area (Ae = 0.47 ± 0.01 cm²).
• Reduced bovine serum albumin (BSA) adsorption confirmed enhanced surface properties.
• Successful one-pot electrochemical digestion of insulin and direct MS analysis were achieved.

Conclusions:

• This work demonstrates the feasibility of carbon SPE-based electrochemical proteolysis for bottom-up proteomics.
• The developed platform offers a miniaturized, inexpensive, and chemical-free alternative for protein digestion.
• This approach is compatible with direct MS detection, streamlining proteomic workflows.