Pseudotrypsin: A Little-Known Trypsin Proteoform.

Area of Science:

• Proteomics
• Enzymology
• Biochemistry

Background:

• Trypsin is a key protease for protein digestion in proteomics.
• Common forms include β-trypsin and α-trypsin.
• Pseudotrypsin (ψ-trypsin) is a three-chain variant with altered properties.

Purpose of the Study:

• To analyze the performance of bovine pseudotrypsin (ψ-trypsin) in protein sample digestion.
• To compare ψ-trypsin's cleavage characteristics with standard trypsin digestion.

Main Methods:

• Experimental analysis using artificial substrates, peptides, and protein standards.
• Ion-exchange chromatography for ψ-trypsin isolation.
• Digestion of protein samples with varying complexity using ψ-trypsin.

Main Results:

• Pseudotrypsin (ψ-trypsin) predominantly cleaves at characteristic trypsin sites.
• ψ-trypsin exhibits increased non-specific cleavages (e.g., after Tyr and Phe) and more missed cleavages compared to standard trypsin.
• ψ-trypsin shows preferential cleavage after basic residues and enhanced side specificity.

Conclusions:

• Pseudotrypsin (ψ-trypsin) provides valuable proteomic information complementary to standard trypsin digestion.
• Its unique cleavage profile can reveal data missed by strictly specific trypsin protocols.
• Understanding ψ-trypsin's performance is crucial for optimizing complex proteomic analyses.