ProteoSure™ Modified Trypsin, Autolysis-Resistant, for proteomic sequencing and mass spectrometry analysis

Trypsin is an essential serine protease that specifically hydrolyzes peptide bonds at the carboxyl side of lysine and arginine residues.  It has been widely used in mass spectrometry-based protein analysis and proteomics studies due to its high proteolytic activity and cleavage specificity. However, the native trypsin, which is referred to as β-trypsin, is also subject to autolytic digestion. Autolysis results in an enzyme form as α-trypsin that shows reduced enzyme stability and efficiency. This form of enzyme can be further autolytically degraded to generate pseudotrypsin (also known as ψ-trypsin), which exhibits a broadened specificity including a chymotrypsin-like activity. The stability, selectivity, and activeness of this enzyme is critical for reproducible protein digestion and mass spectrometry-based protein identification.

ProteoSure™ Sequencing Grade Trypsin is a highly purified porcine trypsin variant that has been genetically improved, chemically modified for the highest autolysis-resistance and maximum specific tryptic activity in proteomic applications.  

ProteoSure™ recombinant porcine trypsin variant is expressed and purified from E.coli in a strictly controlled system. The enzyme is free of contaminating protease activities, such as chymotrypsin. Therefore, it does not need to be treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK), as distinct from other sequencing grade trypsin proteases that are derived from animal pancreatic extracts and have been treated with (TPCK) to remove chymotrypsin activity.

ProteoSure™ recombinant porcine trypsin variant is genetically engineered and further modified by reductive methylation to achieve even greater resistance to autolytic digestion, in comparison to other modified native trypsin proteases (see below Figure 1).

Trypsin can be used in combination with Lysyl Endopeptidase (Lys-C), which has been shown to improve the sequence coverage in peptide mapping.

Product highlights

• Less wait time for sample preparation: Highly active, specific enzyme activity enables fast digestion
• Unambiguous mass spectrometry spectral analysis:

Free of non-tryptic protease contaminants

Extremely low autolysis

High activity between pH 7.0 to pH 8.0, adaptable to a digestion protocol with non-basic pH under non-elevated temperature

• High confidence data interpretation enabled by minimizing the noise from those unwanted non-tryptic peptide fragments, as well as by avoiding the occurrence of artificial deamidations.
• Better coverage in peptide mapping after rapid digestion
• Versatile for wide range of proteomics applications

Applications

It is suitable for in-solution or in-gel protein digestion in applications:

• Peptide sequencing and tryptic mapping
• Protein identification by peptide mass fingerprinting or MS/MS spectral matching.
• Protein-structure studies

Technical notes

ProteoSure™ recombinant porcine trypsin variant possesses mutation that is dedicatedly optimized for greater autolysis-resistance. Most commonly used MS grade Trypsin proteases are modified from native Trypsin that are derived from porcine or bovine pancreatic extracts. Although modification by reductive methylation significantly reduces autolysis, autolytic products of modified Trypsin are commonly found in digests and show as characteristic peaks in the chromatogram of HPLC. It has been also suggested that these autolytic products are present during the purification process from the pancreas  prior to modification.

        By introducing mutations to the porcine trypsin sequence, the resultant protease variant has obtained much greater autolysis-resistance after modification by reductive methylation at Lysine residues, in comparison with the modified native protease. Shown in Figure 1, after enzymatic digestion incubated with ProteoSure Sequencing Grade Modified Tryspin, the characteristic peaks of the autolysed trypsin fragments are not detectable by RP-HPLC.

Figure 1. Autolysis of the modified native porcine trypsin from Manufacturer P and Marvelgent’s ProteoSure™ Sequencing Grade Modified Trypsin. The two proteomic grade trypsin proteases were diluted to 0.05 mg/mL in 50 mM NH4HCO3 (pH 8.0), and then incubated at 37°C. After 3 hours incubation, the protein samples were analyzed by RP-HPLC. In comparison with the protein prior to incubation, the modified trypsin from Manufacturer P has the protein peak shifted from the original position after 3 hour incubation at 37°C, suggesting that autolysis of trypsin occurred and it resulted in smaller fragments of trypsin. However, ProteoSure™ Modified Recombinant Trypsin showed a peak at the same position before and after 3 hour incubation at 37°C, suggesting that the enzyme remained intact. 

Instructions to use

1. Dilute the product in 50mM HAc when needed. Immediately before use, dilute it with 50mM NH₄HCO₃ or a pH7-8 buffer. It is recommended to add 1mM CaCl₂ to the reaction. The ratio of Trypsin to the protein sample should be between 1:20 and 1:100 (w/w). Keep the conditions within the optimum pH of Trypsin (pH7.8 - 8.7).
2. Thaw a frozen aliquot of Trypsin solution at room temperature, and gently mix before use. 
3. When 0.05 mg/ml modified recombinant trypsin is used in buffer 50mM NH₄HCO₃, it retains greater than 95% activity after 3 hours incubation at 37°C. If longer digestion is desired, such as 20 hour incubation at 37°C, it is recommended to include 1mM CaCl₂ to the reaction.

Recommended storage

1. The solution should be stored at -70°C or below. It is stable within 24 months.
2. Greater than 95% enzymatic activity retains after 5 repeated freezing-and-thawing cycles.

Product Details

Documents

Product Information Sheet

🗎     Modified, Engineered Porcine Trypsin, Sequencing-Grade, Autolysis-resistant

Technical Note

🗎     Conversion of Enzyme Activity Measurement Units

Marvelgent Biosciences Inc.

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