An N terminomics toolbox combining 2-pyridinecarboxaldehyde probes and click chemistry for profiling protease specificity.

Proteomic profiling of protease-generated N termini provides key insights into protease function and specificity. However, current technologies have sequence limitations or require specialized synthetic reagents for N-terminal peptide isolation. Here, we introduce an N terminomics toolbox that combines selective N-terminal biotinylation using 2-pyridinecarboxaldehyde (2PCA) reagents with chemically cleavable linkers to enable efficient enrichment of protein N termini. By incorporating a commercially available alkyne-modified 2PCA in combination with Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), our strategy eliminates the need for chemical synthesis of N-terminal probes. Using these reagents, we developed PICS2 (Proteomic Identification of Cleavage Sites with 2PCA) to profile the specificity of subtilisin/kexin-type proprotein convertases (PCSKs). We also implemented CHOPPER (chemical enrichment of protease substrates with purchasable, elutable reagents) for global sequencing of apoptotic proteolytic cleavage sites. Based on their broad applicability and ease of implementation, PICS2 and CHOPPER are useful tools that will advance our understanding of protease biology.

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 ⍺-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 through 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, an enzymatic modification reagent, and 2-pyridinecarboxaldehyde (2PCA), a chemical modification reagent, 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. Although we detail the steps for profiling the specificity of 2PCA and subtiligase in Escherichia coli and human cells, these protocols can easily be adapted to alternative proteome sources and other N-terminal peptide labeling reagents. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of N-terminally diverse proteome-derived peptide libraries from E. coli Alternate Protocol: Generation of N-terminally diverse proteome-derived peptide libraries from human cells Basic Protocol 2: Characterizing the specificity of 2-pyridinecarboxaldehyde using proteome-derived peptide libraries Basic Protocol 3: Characterizing the specificity of subtiligase using proteome-derived peptide libraries.