Details

Autor(en) / Beteiligte

• Bridge, Haley N.
• Leiter, William
• Frazier, Clara L.
• Weeks, Amy M.

Titel

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

Ist Teil von

• Cell chemical biology, 2024-03, Vol.31 (3), p.534-549.e8

Ort / Verlag

Elsevier Ltd

Erscheinungsjahr

2024

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• 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. [Display omitted] •2PCA is a broad-specificity N-terminal probe•2PCA probes rapidly define protease sequence specificity•Clickable 2PCA probes enable proteomic profiling of protease sites•2PCA-based enrichment is highly complementary to other N terminomics technologies Bridge et al. introduce an N terminomics strategy that combines 2-pyridinecarboxaldehyde (2PCA) probes, click chemistry, and cleavable linkers for enrichment of protein N termini. This technology enables protease sequence and substrate specificity profiling on a proteome scale using commercially available reagents.