An N terminomics toolbox combining 2-pyridine carboxaldehyde probes and click chemistry for profiling protease specificity

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The paper invents an expanded toolbox of reagents based on 2-pyridinecarboxaldehyde (2PCA) for profiling protease substrates and specificity using chemoproteomic methods. The authors performed deep profiling of 2PCA specificity using proteome-derived peptide libraries, finding it has high N-terminal selectivity with little intrinsic sequence bias beyond a requirement for a backbone amide at position 2. Then optimized conditions for efficient 2PCA modification of proteomic samples. Designing a biotin-disulfide-2PCA probe to enable “catch and release” enrichment of 2PCA-labeled N-terminal peptides.

With this, the work showed commercially available alkyne-2PCA can be used with click chemistry for modular N-terminal enrichment without probe synthesis. Developing PICS2 method for profiling protease specificity in proteome peptide libraries using 2PCA to block N-termini then enrich neo-N-termini. Then applying PICS2 to profile specificity of furin and other subtilisin/kexin-type proprotein convertases. And introduced method called CHOPPER using alkyne-2PCA for enrichment of protease substrates from cell lysates. Identifying 112 new caspase cleavage sites in apoptosis using CHOPPER. This 2PCA toolkit enables a broad applications in protease biology research by providing selective N-terminal labeling reagents that are easy to implement.

https://www.cell.com/cell-chemical-biology/pdf/S2451-9456(23)00328-8.pdf