Sequencing Grade Modified Trypsin

What is Reductive Methylation, and How Does It Make Trypsin More Stable?

Reductive methylation modifies the lysine residues on trypsin itself, blocking the enzyme’s self-cleavage sites without affecting its activity toward substrate proteins. The result is a stable, highly active enzyme that resists autolysis throughout extended digestion incubations.

Trypsin cleaves at lysine and arginine residues—including those within its own structure. Reductive methylation adds methyl groups to the ε-amino groups of trypsin’s internal lysine residues, making them resistant to tryptic cleavage. This chemical modification does not alter the enzyme’s active site or its recognition of substrate Lys and Arg residues, so catalytic activity is preserved. The practical result: very few autolytic fragments appear in your digest. Because the enzyme cannot cleave itself, the peptide mixture you analyze reflects only your target protein—not breakdown products of the protease.

What Is TPCK Treatment, and Why Does It Matter for Proteomics?

Tosyl phenylalanyl chloromethyl ketone (TPCK) irreversibly inactivates chymotrypsin by blocking its active site. TPCK-treated trypsin preparations are free of chymotryptic activity, which eliminates nonspecific cleavages at aromatic and large hydrophobic residues that would otherwise generate unexpected peptides.

Commercially available porcine trypsin can contain trace chymotrypsin contamination co-purified from pancreatic extracts. Even small amounts of chymotrypsin can cause cleavage at Tyr, Phe, Trp and Leu residues—sites that should be intact in a tryptic digest. These nonspecific cleavages create peptides that don’t match expected tryptic fragments, increasing missed matches and reducing identification confidence. Sequencing Grade Modified Trypsin is treated with TPCK and then purified by affinity chromatography, providing a preparation with high tryptic specificity and no detectable chymotryptic activity.

Sequencing-Grade Modified Trypsin is only tested for stability and sequence specificity. Promega cannot guarantee that Sequencing-Grade Modified Trypsin will perform if used for mass spectrometry applications. Trypsin Gold and Trypsin Platinum are qualified for mass spectrometry applications. Each lot of the Trypsin Gold, Mass Spectrometry Grade (V5280) is qualified by mass spectrometry to ensure compatibility with your applications/instrumentation. For experiments requiring maximum digestion efficiency with the fewest missed cleavages—such as deep proteome coverage or large-scale quantitative datasets—consider Trypsin Platinum, Mass Spectrometry Grade (VA9000), which offers further optimized specifications for demanding mass spectrometry applications. See the product comparison table.

Which Promega Trypsin Is Right for My Proteomics Workflow?

Promega offers sequencing-grade and mass spectrometry (MS)-grade trypsin options optimized for different applications and levels of digest complexity. Sequencing Grade Modified Trypsin is a proven, versatile choice for most protein sequencing. Trypsin Gold is qualified for use in MS applications. Trypsin Platinum provides maximum specificity for demanding quantitative or low-abundance MS applications.