Proteome-derived peptide libraries for deep specificity profiling of N-terminal modification reagents

Protein and peptide N termini are important targets for selective modification with chemoproteomics reagents and bioconjugation tools. The N-terminal α-amine occurs only once in each polypeptide chain, making it an attractive target for protein bioconjugation. In cells, new N termini can be generated by proteolytic cleavage and captured by N-terminal modification reagents that enable proteome-wide identification of protease substrates with tandem mass spectrometry (LC-MS/MS). An understanding of the N-terminal sequence specificity of the modification reagents is critical for each of these applications. Proteome-derived peptide libraries in combination with LC-MS/MS are powerful tools for profiling the sequence specificity of N-terminal modification reagents. These libraries are highly diverse, and LC-MS/MS enables analysis of the modification efficiencies of tens of thousands of sequences in a single experiment. Proteome-derived peptide libraries are a powerful tool for profiling the sequence specificities of enzymatic and chemical peptide labeling reagents. Subtiligase and 2-pyridinecarboxaldehyde (2PCA) are two reagents that have been developed for selective N-terminal peptide modification and can be studied using proteome derived peptide libraries. This protocol outlines the steps for generating N-terminally diverse proteome-derived peptide libraries and for applying these libraries to profile the specificity of N-terminal modification reagents. We detail the steps for profiling the specificity of 2-pyridinecarboxaldehyde, a chemical modification reagent, and subtiligase, an enzymatic modification reagent. These protocols can easily be adapted to alternative proteome sources and other N-terminal peptide labeling reagents.