In proteomics applications, the choice of proteolytic enzyme is critical for achieving confident protein identification and comprehensive proteome coverage. High enzymatic efficiency is essential to digest complex protein mixtures into peptides suitable for LC–MS analysis. This increases peptide yield, improves sequence coverage, and enhances the likelihood of identifying low-abundance proteins. In this context, a high number of unique peptides - particularly peptides generated without any missed cleavages - is a key indicator of enzyme performance, as it directly contributes to increased confidence in consistent protein identification and quantification.
 
To evaluate digestion performance in a proteomics setting, human HeLa cell lysates were digested using LysCERATOR and two alternative Lys-C enzymes (Alternative Lys-C A and Alternative Lys-C B, Fig. 1a) and the numbers of unique peptides were compared. Following digestion, peptides were cleaned up using protein aggregation capture (PAC) on magnetic beads and analyzed by LC–MS. LysCERATOR generated both the highest number of unique peptides and the highest proportion of peptides containing zero missed cleavages of the three Lys-C enzymes tested. The higher proportion of fully cleaved peptides using LysCERATOR indicates superior digestion efficiency on complex biological samples, resulting in a more informative and interpretable peptide pool compared to the alternative enzymes.
 
Enzyme specificity is another critical parameter in proteomics workflows. A high level of amino acid specificity ensures predictable cleavage patterns and reduces the number of nonspecific peptides. This, in turn, minimizes spectral complexity and decreases ambiguity during peptide-to-spectrum matching. Three different batches of LysCERATOR were evaluated, and the lysine specificity was consistently high, at approximately 97–98% (Fig. 1b). The observed batch-to-batch reproducibility highlights the robustness and reliability of LysCERATOR for proteomics workflows.
 
LysCERATOR combines very high lysine specificity with superior digestion efficiency, making it well suited for complex proteomics workflows. These features result in cleaner spectra, greater confidence in protein identification, and more robust data analysis – particularly for highly complex samples with inherently large numbers of potential peptide assignments.

Figure 1. a) Comparison of Lys-C digestion of HeLa cells. HeLa cell lysate was digested in triplicate, with LysCERATOR and two alternative Lys-C enzymes, at an enzyme to substrate ratio of 1:100 in 50 mM HEPES buffer (pH 8.5) overnight at 37°C. Peptide clean-up was performed with PAC on magnetic beads and data were acquired by LC-MS. Graphs show the number of unique peptides identified grouped by the number of missed cleavages. Error bars show the standard deviation. b) Lysine specificity of LysCERATOR. Three different batches of LysCERATOR were used to digest HeLa cell lysate at an enzyme to substrate ratio of 1:100 in 50 mM HEPES buffer (pH 8.5) overnight at 37°C. Peptide clean-up was performed with PAC on magnetic beads and data were acquired by LC-MS. The amino acid specificity of the different batches of LysCERATOR was determined based on the frequency of the C-terminal amino acid from the identified peptides.

Cleavage at lysine residues followed by proline (K–P sites) is a well-established challenge for Lys-C digestion, often leading to missed cleavages due to proline-induced conformational constraints. To assess how effectively LysCERATOR overcomes this limitation, its performance at K–P sites was evaluated in detail and compared with two alternative, commercially available Lys-C enzymes - one non-recombinant and one recombinant.

Digestion efficiency was assessed using trastuzumab. Figure 4 shows the relative intensities of two peptides derived from the light chain N-terminus: LC aa 1–39, representing the expected cleavage, and LC aa 1–42, which contains a missed cleavage at the K39/P40 site. After 2 hours of digestion with LysCERATOR, the fully cleaved peptide (LC aa 1–39) was the more abundant species, while digestion with the non-recombinant alternative Lys-C predominantly produced the miscleaved peptide (LC aa 1–42).

Following overnight digestion with LysCERATOR, there was a near-complete digestion at the K-P site while, in contrast, a substantial amount of the miscleaved peptide remained after digestion with the non-recombinant alternative Lys-C. Digestion with the recombinant alternative Lys-C resulted in low overall signal intensity for both peptides, with the most prominent species being a peptide containing two missed cleavages (LC aa 1–45 – not shown).

Collectively, these results demonstrate that LysCERATOR exhibits superior digestion efficiency at challenging K–P sites compared with alternative Lys-C enzymes, resulting in fewer missed cleavages and more complete proteolytic processing.

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 assignment 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 are essential for defining critical quality attributes (CQAs).

To investigate potential autoproteolytic tendencies of LysCERATOR, the enzyme was incubated overnight in reaction buffer (0.1 M Tris-HCl, pH 8.0) at 37°C and analyzed by RP-LC–MS (Fig. 6). A freshly prepared enzyme sample was analyzed in parallel as a control. No generation of LysCERATOR peptides was observed following incubation of the enzyme alone, compared to the control, consistent with very low or undetectable levels of LysCERATOR autoproteolysis under these conditions.

The absence of detectable autoproteolysis demonstrates the effectiveness of LysCERATOR for peptide mapping applications, particularly for extended or overnight digestions commonly used in antibody characterization. By minimizing enzyme-derived background signals and maintaining consistent proteolytic activity, low levels of autoproteolysis contribute to cleaner mass spectra, more confident peptide identification, and improved robustness of peptide mapping methods throughout antibody drug development.