Minimal Autoproteolysis using TrypsINATOR™

Performance Activity

Robust digestion performance with limited autoproteolysis, enabling cleaner LC–MS data and more confident peptide identification.

A low level of autoproteolysis is an important quality attribute of proteolytic enzymes used for peptide mapping of antibody candidates in drug development. Autoproteolysis – self-digestion of the enzyme – can negatively affect analytical performance in two important ways. Firstly, it may reduce the effective enzyme activity during longer digestion times, leading to incomplete digestion and increased variability. Secondly, peptides generated from enzyme self-cleavage can co-elute with analyte-derived peptides and introduce contaminating signals during LC–MS analyses, complicating peak assignments and increasing the risk of misidentification. This added ambiguity is particularly problematic in peptide mapping workflows, where confident sequence confirmation and precise localization of post-translational modifications (PTMs) are essential for defining critical quality attributes (CQAs).

 

TrypsINATOR is a recombinantly expressed trypsin, based on the Sus scrofa sequence, and free from undesirable contaminants such as chymotrypsin, which can otherwise impact both the specificity and activity of trypsin. To maximize autoproteolytic resistance and stability, TrypsINATOR is reductively methylated at lysine residues.

Recombinantly expressed and reductively methylated trypsin to maximize autoproteolytic resistance

To determine the level of autoproteolysis, trastuzumab was digested using both TrypsINATOR and TrypsINATOR LysCERATOR Mix for 2 hours, and the total intensity of autoproteolytic peptides was compared to the total intensity of trastuzumab-derived peptides. TrypsINATOR autoproteolysis was determined to be considerably lower than that of the alternative products (Fig. 1a), and TrypsINATOR LysCERATOR Mix autoproteolysis appeared to be similar to competitor mix A, and considerably lower than competitor mix B (Fig. 1b). The limited observed autoproteolysis qualifies both TrypsINATOR and TrypsINATOR LysCERATOR Mix as effective tools for peptide mapping, providing cleaner mass spectra, more confident peptide identification and improved robustness.

Minimal autoproteolysis for TrypsINATOR and TrypsINATOR LysCERATOR Mix

To further verify the stability of TrypsINATOR, the enzyme was incubated in 50 mM Tris-HCl, pH 8.0 at 37°C for 2 hours and analyzed by RP-LC-MS/MS. A freshly prepared enzyme sample was analyzed in parallel and used as a control. Following incubation of the enzyme alone, no generation of TrypsINATOR peptides was observed compared to the control, consistent with very low or undetectable levels of TrypsINATOR autoproteolysis under these conditions (Fig. 1c).

No detectable autoproteolysis of TrypsINATOR in reaction buffer

Figure 1. Minimal autoproteolysis of TrypsINATOR and TrypsINATOR LysCERATOR Mix. Relative autoproteolytic intensity of a) TrypsINATOR and b) TrypsINATOR LysCERATOR Mix, compared to alternative products, following incubation of trastuzumab at 37°C for 2 hours. The relative autoproteolytic intensity is calculated as total intensity of autoproteolytic peptides relative to total intensity of trastuzumab-derived peptides. c) Total ion chromatogram of TrypsINATOR before and after incubation at 37°C for 2 hours in 50 mM Tris-HCl, pH 8.0.

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