Tryp-N™ is a thermophilic metalloprotease with N-terminal specificity for arginine and lysine engineered at Cold Spring Harbor Laboratory (CSHL). Tryp-N™ provides clarified spectra and greater sensitivity by yielding peptides in which the basic centers of peptides – the amino terminus and side chains of K/R – are located together to produce b-ion dominated spectra. Tryp-N has been demonstrated to yield simplified MS/MS fragmentation and have the best proteolytic activity.¹ Tryp-N spectra provide the complimentary ion series to compliment trypsin, allowing you to more easily find and specifically localize PTMs. PTM identification and localization of PTMs, such as phosphorylation in YST clusters, and de novo sequencing. Additionally, as ion current remains on b-ions during fragmentation of MRM assays, rather than becoming diluted between two charged termini in tryptic peptides, sensitivity is often enhanced.
As a thermophilic enzyme, digestions can be completed in minutes to hours, not over night. Digestions can be easily control digestion with the addition of EDTA for reproducible partial digests and increased sequence coverage. Tryp-N™ cleavage is unaffected by proline or lysine methylation. At higher temperatures – Topt is 65 °C – proteins are denatured, assisting in the analysis of difficult-to-digest proteins. Tryp-N™ is highly stable across a wide variety of experimental conditions including pH (5-11), buffers, ionic strength and detergents. At higher temperatures, endogenous enzymes are denatured and inactive, a useful train to deal with pesky phosphatases and proteases.
The speed, specificity, ease of use and overall effectiveness of Tryp-N protease, combined with its ability to provide more interpretable spectra make it a valuable component of the proteomics toolkit.
¹Wilson JP, Ipsaro JJ, Del Giudice SN, Turna NS, Gauss CM, Dusenbury KH, Marquart K, Rivera KD, Pappin DJ. Tryp-N: A thermostable protease for the production of N-terminal argininyl and lysinyl peptides. Journal of Proteome Research. 2020 Mar 6;19(4):1459-69.