Chemoselective protein and peptide immobilization on biosensor surfaces

Area of Science:

• Biochemistry
• Analytical Chemistry
• Surface Science

Background:

• Site-specific immobilization of biomolecules on sensor surfaces is crucial for accurate bioanalytical measurements.
• Conventional protein immobilization methods often lead to heterogeneous ligand presentation, potentially causing data variability in surface plasmon resonance (SPR) assays.
• Heterogeneous immobilization can result in conflicting data compared to solution-based studies.

Purpose of the Study:

• To present two versatile bioconjugation strategies for chemoselective immobilization of peptides and proteins.
• To address the challenge of achieving uniform ligand presentation on sensor surfaces for enhanced bioanalytical applications.
• To provide adaptable protocols for SPR and other protein-functionalized surface applications.

Main Methods:

• Utilizing native chemical ligation for C-terminus specific protein immobilization.
• Employing oxime ligation for N-terminus specific peptide and protein immobilization.
• Adapting protocols for Biacore SPR instruments, with considerations for other SPR systems.

Main Results:

• Demonstrated chemoselective immobilization via either N- or C-termini.
• Enabled site-specific attachment of proteins and peptides, ensuring consistent orientation.
• Established generic bioconjugation methods applicable across different SPR platforms.

Conclusions:

• Developed complementary methods for controlled protein and peptide immobilization on sensor surfaces.
• These techniques offer improved ligand presentation, enhancing the reliability of SPR and other surface-based bioassays.
• The described bioconjugation strategies provide a foundation for advancing bioanalytical applications requiring functionalized surfaces.