New England Biolabs, P8119S, Trypsin-ultra™, Mass Spectrometry Grade (Recombinant)

Trypsin-ultra, Mass Spectrometry Grade (Recombinant) is a serine protease recombinantly expressed in Related Categories Proteases,, Proteome Analysis Applications Protein Analysis Tools,, Glycobiology & Proteomics,, Glycoprotein Analysis Specification Reaction Conditions Buffer of choice, pH 7-9 Trypsin:substrate ratio from 1:10 to 1:200 Incubate at 37°C Molecular Weight Apparent: 22.6 kDa FAQ Q: What is the difference between Trypsin-ultra™, Mass Spectrometry Grade (Recombinant) (NEB #P8119), and Trypsin-ultra, Mass Spectrometry Grade (NEB #P8101)? A: Trypsin-ultra, Mass Spectrometry Grade (Recombinant) (NEB #P8119) is recombinantly expressed porcine trypsin manufactured without the use of any animal-derived components. It is mutagenized and further chemically modified via methylation to make the enzyme exceptionally resistant to autolysis. In contrast, Trypsin-ultra, Mass Spectrometry Grade (NEB #P8101), is isolated and purified from bovine pancreas, treated with TPCK to inactivate the copurified chymotrypsin activity, and additionally acetylated to reduce autolysis. Because it is cloned and expressed in E. coli, Trypsin-ultra, Mass Spectrometry Grade (Recombinant) is naturally free of contaminating chymotrypsin and has enhanced autolysis resistance. These properties improve chromatogram quality and mass spectrometry data, resulting in high-confidence quantitation and identification of peptides. Trypsin-ultra, Mass Spectrometry Grade (Recombinant) is provided in a user-friendly, freeze-dried format, and is stable for 24 months. Q: What are the advantages of recombinant trypsin (NEB #P8119)? A: The purity and absence of contaminating protease activity are crucial for the data quality of peptide mapping and bottom-up proteomics experiments. Trypsin is typically derived from animal sources, e.g., porcine pancreas. Other digestive proteases, such as chymotrypsin, are often co-purified along with trypsin. Trypsin-ultra™, Mass Spectrometry Grade (Recombinant) is inherently free of contamination. Moreover, because it is engineered to reduce autolysis, trypsin-derived peptides are absent from the spectra. Together, its purity and autolysis resistance result in specific proteolysis, reduced spectra complexity, and more confident protein identification and quantification, thereby improving data analysis and reporting in a regulated environment. Q: Why is autolysis-resistance important? What are the features that make Trypsin-ultra™, Mass Spectrometry Grade (Recombinant) (NEB #P8119) autolysis resistant? A: Autolysis of trypsin can produce several undesirable outcomes. Firstly, autolysis reduces trypsin activity, leading to incomplete digestion and missed cleavages. Secondly, an intermediate autolysis product, ψ-trypsin, has relaxed specificity and cleaves amino acids other than lysine and arginine, exhibiting chymotrypsin-like activity. Thirdly, autolysis can also make aliquoting, storage and handling of trypsin challenging. Recombinant trypsin is engineered to retain its enzymatic properties and is further methylated to make it exceptionally resistant to autolysis. This leads to better data quality, enzyme stability, and easy handling. Q: How should I aliquot and store Trypsin-ultra™, Mass Spectrometry Grade (Recombinant) (NEB #P8119)? A: For optimal long-term stability, reconstitute Trypsin-ultra, Recombinant, Mass Spectrometry Grade (Recombinant) with high-purity water to 0.1 to 1 µg/µL, as needed, and store the stock solution at -20 °C for up to 6 months. Enzyme activity can be retained for five freeze-thaw cycles. Q: Which reaction buffer can be used for trypsin digestion? A: Trypsin is compatible with many buffer types, with the optimal pH between 8-9. Typical reaction buffers are 50 mM Tris-HCl, pH 8.0, or 50 mM ammonium bicarbonate, pH 7.8. Calcium ions, although not necessary for its activity, can stabilize trypsin, especially in extended digestion conditions. A concentration of 1 - 10 mM is often used. The selection of the digestion buffer also depends on how the protein sample is collected or prepared, and if the digestion is immediately followed by mass spectrometry analysis. Generally, ammonium bicarbonate buffer is considered the most mass spectrometry-friendly buffer system, as both the anions and cations are volatile. Q: What is the optimal ratio of trypsin to protein for digestion? A: The trypsin:protein ratio is often between 1:20 and 1:100, with 1:50 commonly recommended, resulting in efficient digestion without excessive trypsin autolysis. This ratio is often dependent on factors such as the nature and source of the protein sample, the use of an optimal buffer, and the incubation temperature. When using Trypsin-ultra™, Mass Spectrometry Grade (Recombinant) (NEB #P8119) for fast or high-throughput workflows, we have used ratios of 1:5 or 1:10 (without observable artifacts), enabling the digest to be performed in 30 minutes at 37 °C. Q: Is trypsin compatible with detergents? A: Detergents and high concentrations of chaotropic agents, such as SDS, urea, and guanidine hydrochloride, are often used to denature protein samples before protease treatment. However, high concentrations of chaotropic agents and ionic detergents (such as SDS at > 0.1%) inhibit trypsin. Denatured samples need to be buffer-exchanged or dialyzed into a suitable trypsin reaction buffer before trypsin is added for digestion. Some non-ionic detergents, such as NP-40 and Triton, can be compatible with trypsin digestion at low levels but must be removed from the sample before mass spectrometry analysis. Q: At what temperature should trypsin digestion be performed? A: Most often trypsin digestion is carried out at 37 °C, but trypsin reactions can be incubated at elevated temperatures (50 to 70 °C) for faster proteolysis. In this case, the thermostability of the protein sample and its post-translational modifications need to be considered (i.e. increased rate of asparagine deamidation). Q: How do I desalt peptides after trypsin digestion? A: After trypsin digestion and before mass spectrometric analysis, peptide samples can be cleaned to remove contaminants that interfere with mass spectrometry. This can be accomplished using size-exclusion columns, solid-phase extraction (SPE) spin columns, or cartridges. Q: Can other proteases be used along with trypsin? A: Double protease digestions, performed sequentially or simultaneously, are sometimes used to enhance site-specific cleavage or to achieve maximal coverage of protein sequences. The other protease can be either complementary (e.g., Endoproteinase GluC, NEB #P8100, or α-Lytic Protease, NEB #P8113) or supplementary (e.g., Endoproteinase LysC, NEB #P8109).