Hydrazide reactive peptide tags for site-specific protein labeling

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• Site-specific protein labeling (SSPL) is crucial for real-time protein detection within living cells.
• Existing SSPL methods often rely on bioorthogonal reagents and complementary peptides.

Purpose of the Study:

• To develop novel site-specific protein labeling (SSPL) reactions targeting specific peptides.
• To enable real-time detection of proteins inside living cells.
• To establish methods for identifying peptides reactive with bioorthogonal reagents.

Main Methods:

• Selection of hydrazide reactive (HyRe) peptides from phage-displayed libraries using reaction-based selections.
• Utilizing washes at varying pH and sodium cyanoborohydride (NaCNBH3) treatment for peptide selection.
• Assessing peptide-hydrazide interactions via colorimetric assays and protein fusions (T4 lysozyme).
• Characterizing covalent adducts using MALDI-TOF MS and MS/MS analysis.

Main Results:

• Demonstrated specific, covalent labeling of a peptide-lysozyme fusion protein in bacterial lysates using HyRe peptides.
• Identified reaction with the N-terminal His of a HyRe tag at neutral pH, and an alternative adduct on Ser6 at low pH (<6.0).
• Confirmed formation of covalent adducts with distinct hydrazide probes via mass spectrometry.

Conclusions:

• The novel bimolecular reaction offers a potential tool for in vivo protein labeling and bioconjugate synthesis.
• Optimization of this reaction could significantly advance protein labeling technologies.
• The selection and screening methods are broadly applicable for identifying peptides for other SSPL reactions.