Elucidating Tertiary Structures of Affibody in Vacuo Using Genetic Code Expansion and FRIPS Mass Spectrometry

Area of Science:

• Structural Biology
• Mass Spectrometry
• Biochemistry

Background:

• Protein structure elucidation is crucial for understanding biological function.
• Gas-phase protein analysis offers unique insights into molecular conformations.
• Free radical-initiated peptide sequencing (FRIPS) mass spectrometry (MS) is a powerful tool for gas-phase protein fragmentation.

Purpose of the Study:

• To develop a novel method for in vacuo protein structural analysis.
• To combine FRIPS MS with genetic code expansion (GCE) technology.
• To investigate protein folding and tertiary structure using residue-specific fragmentation.

Main Methods:

• Incorporation of unnatural amino acids (UAAs) as radical precursors into an Affibody protein at six distinct sites.
• Utilizing FRIPS MS to induce radical-directed fragmentation.
• Analyzing fragmentation patterns to infer radical transfer pathways and proximity within the tertiary structure.

Main Results:

• The Affibody protein retained a folded conformation in the lowest charge state (+5), similar to its native structure.
• Significant radical-directed fragmentations were observed via both "through-sequence" and "through-space" mechanisms.
• Demonstrated the capability of FRIPS MS to provide residue-specific structural information.

Conclusions:

• FRIPS MS combined with GCE is a viable approach for gas-phase protein structural analysis.
• The method offers residue-specific insights into protein folding and tertiary structure.
• This technique advances the detailed structural characterization of proteins in vacuo.