Photoredox-Catalyzed Decarboxylative C-Terminal Differentiation for Bulk- and Single-Molecule Proteomics

Area of Science:

• Proteomics and Analytical Chemistry
• Biochemistry and Molecular Biology

Background:

• Selective labeling of peptides is crucial for proteomics technologies like single-molecule fluorosequencing.
• Peptide fragments universally possess N-terminal amine and C-terminal carboxylic acid groups, making them ideal targets for global labeling.

Purpose of the Study:

• To introduce and validate a new method for site-specific C-terminal peptide labeling using photocatalyzed decarboxylative alkylation.
• To establish a broadly applicable labeling technology for diverse proteomes.

Main Methods:

• Application of photocatalyzed C-terminal decarboxylative alkylation on peptides from enzymatic digests of bovine serum albumin, yeast, and human cell extracts.
• Characterization of labeling efficiencies and amino acid residue proclivity using two analytical approaches.
• Combination of C-terminal labeling with orthogonal carboxylic acid labeling for fluorosequencing.

Main Results:

• Demonstrated successful site-specific labeling of C-terminal residues in complex peptide mixtures.
• Achieved C-terminal labeling efficiencies exceeding 50% across complete proteomes.
• Identified histidine and tryptophan as disfavored residues for decarboxylative labeling.

Conclusions:

• Photocatalyzed C-terminal decarboxylative alkylation is a viable global labeling strategy for mass spectrometry and single-molecule protein sequencing.
• The developed method enables broad application across various proteomes.
• Integration with other labeling techniques creates a new platform for advanced protein analysis and sequencing.