ProtiFi Tryp-N™ Sequencing Grade Kit contains a thermophilic metalloprotease designed for enzymatic protein digestion prior to LC-MS/MS analysis. Tryp-N™ cleaves N-terminally to lysine (K) and arginine (R), providing complementary specificity to trypsin and generating peptides that produce b-ion-dominated MS/MS spectra. This makes it particularly useful for structural protein characterization, post-translational modification localization, de novo sequencing, and MRM workflows.
Tryp-N™ operates at elevated temperatures of 55–65 °C, with an optimum temperature of approximately 65 °C. Elevated-temperature digestion helps denature difficult proteins while inactivating many endogenous proteases and phosphatases, allowing digestion to be completed in minutes to a few hours rather than overnight.
Tryp-N™ Cleavage Specificity
Unlike trypsin, which cleaves C-terminally to lysine and arginine, Tryp-N™ cleaves on the N-terminal side of K and R residues.
The resulting peptide charge distribution favors b-ion-dominated fragmentation, providing a complementary ion series to conventional tryptic peptides. This can simplify MS/MS interpretation and improve sequence or PTM localization in selected workflows.
Rapid High-Temperature Digestion
Tryp-N™ is thermophilic and performs digestion at 55–65 °C. The current standard protocol uses:
- 55 °C
- 3 h digestion
- no shaking.
Its optimum temperature is approximately 65 °C, while robust activity is maintained at 55 °C, a temperature that can reduce thermal precipitation problems for some samples.
EDTA-Controlled Proteolysis
As a metalloprotease, Tryp-N™ activity can be controlled through metal chelation with EDTA. This enables reproducible termination or controlled partial digestion.
For in-solution digestion, the current protocol specifies 2.5–5 mM final EDTA as an optional digestion-stop condition.
Technical Specifications
- Enzyme Type: Thermophilic metalloprotease
- Application: Protein digestion prior to MS
- Cleavage Specificity: N-terminal to lysine and arginine
- MS/MS Characteristic: b-ion-dominated fragmentation
- Optimum Temperature: approximately 65 °C
- Working Temperature: 55–65 °C
- Standard Protocol Digestion: 55 °C for 3 h
- Stability Range: approximately pH 5–11
- Supplied Enzyme Concentration: 0.5 mg/mL
- Recommended Enzyme-to-Protein Ratio: 1:50 (w/w)
- Typical Sample Amount: 1–100 µg total protein
- Activity Control: EDTA
- Optional Digestion Stop: 2.5–5 mM EDTA
- Compatible Workflows: in-solution digestion, S-Trap™
- Downstream Techniques: LC-MS, LC-MS/MS, MRM
Available Kit Sizes
PN K001-TRYPN-0010uG
- Tryp-N™ Sequencing Grade Enzyme: 10 µg
- Tryp-N™ Digestion Solution: 250 µL
PN K001-TRYPN-0020uG
- Tryp-N™ Sequencing Grade Enzyme: 20 µg
- Tryp-N™ Digestion Solution: 500 µL
PN K001-TRYPN-0100uG
- Tryp-N™ Sequencing Grade Enzyme: 100 µg
- Tryp-N™ Digestion Solution: 2 × 1.5 mL
The Tryp-N™ Sequencing Grade Enzyme is supplied in 50 mM trimethylammonium acetate, 2 mM CaCl₂, and 100 µM MnCl₂ in LC-MS-grade water.
The Tryp-N™ Digestion Solution contains 100 mM trimethylammonium acetate, pH 7.4 at 55 °C, 4 mM CaCl₂, and 200 µM MnCl₂. The product is shipped on dry ice.
Typical Applications
- Bottom-up proteomics
- LC-MS/MS proteomics
- Complementary digestion to trypsin
- PTM localization
- Phosphoproteomics
- De novo sequencing
- MRM / targeted proteomics
- Improved protein sequence coverage
- Difficult-to-digest proteins
- Controlled / limited proteolysis
- S-Trap™ proteomics workflows
