Proteome-Derived Peptide Libraries for Deep Specificity Profiling of N-terminal Modification Reagents

Area of Science:

• Biochemistry
• Proteomics
• Chemical Biology

Background:

• Protein N-termini are crucial for selective modification in chemoproteomics and bioconjugation.
• N-terminal modification reagents enable proteome-wide identification of protease substrates via mass spectrometry.
• Understanding reagent specificity is vital for these applications.

Purpose of the Study:

• To outline a protocol for generating N-terminally diverse proteome-derived peptide libraries.
• To apply these libraries for profiling the sequence specificity of N-terminal modification reagents.
• To demonstrate the protocol using 2-pyridinecarboxaldehyde (2PCA) and subtiligase in E. coli and human cells.

Main Methods:

• Generation of N-terminally diverse proteome-derived peptide libraries from prokaryotic (E. coli) and eukaryotic (human) cells.
• Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for high-throughput analysis.
• Profiling the specificity of chemical (2PCA) and enzymatic (subtiligase) N-terminal labeling reagents.

Main Results:

• Established a robust protocol for creating diverse peptide libraries.
• Successfully profiled the sequence specificities of 2PCA and subtiligase.
• Demonstrated the adaptability of the protocol for various proteome sources and labeling reagents.

Conclusions:

• Proteome-derived peptide libraries coupled with LC-MS/MS are powerful tools for assessing N-terminal modification reagent specificity.
• The presented protocols facilitate the characterization of chemical and enzymatic labeling reagents.
• This approach supports advancements in chemoproteomics and bioconjugation strategies.