Improved Digestion Efficiency of TrypsINATOR™ on Human Cell Lysates

Application

Highly specific lysine and arginine digestion with more unique peptides and fewer missed cleavages for confident protein identification in complex proteomics workflows.

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 cell lysates were digested using TrypsINATOR and three commercially available trypsin products. Following digestion, peptides were cleaned up using protein aggregation capture (PAC) on magnetic beads and analyzed by LC-MS. TrypsINATOR generated the highest number of unique peptides, and the highest proportion of peptides containing zero missed cleavages of the four trypsin products tested (Fig. 1a). The higher proportion of fully cleaved peptides using TrypsINATOR indicates superior digestion efficiency on complex biological samples, resulting in a more informative and interpretable peptide pool compared to alternative trypsin products.

TrypsINATOR results in more unique peptides and fewer missed cleavages compared to alternative trypsin products

Lys-C is often added to trypsin for faster and more comprehensive digestion of complex substrates, while maintaining the same amino acid specificity. To evaluate digestion performance in a proteomics setting, human cell lysates were also digested using TrypsINATOR LysCERATOR Mix alongside two alternative Trypsin/Lys-C mixes. TrypsINATOR LysCERATOR Mix generated the highest number of unique peptides, with the highest proportion of peptides containing zero missed cleavages of the three mixes tested (Fig. 1b). The efficiency of TrypsINATOR was boosted by the addition of LysCERATOR, demonstrated by an increase in the proportion of peptides with zero missed cleavages from 81% using TrypsINATOR alone, to 88% using TrypsINATOR LysCERATOR Mix. In addition, there was an increase in the number of unique peptides using the mix compared to TrypsINATOR alone. This illustrates that for a more complete digestion in complex mixtures, TrypsINATOR LysCERATOR Mix is preferred to TrypsINATOR alone.

Addition of Lys-C boosts digestion efficiency in the TrypsINATOR LysCERATOR Mix

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 non-specific peptides. This, in turn, minimizes spectral complexity and decreases ambiguity during peptide-to-spectrum matching. TrypsINATOR was determined to have high specificity for arginine and lysine residues (45% for arginine and 54% for lysine residues; 99% in total) with very little non-specific digestion detected (Fig. 1c).

Highly specific digestion at arginine and lysine residues

Figure 1. TrypsINATOR and TrypsINATOR LysCERATOR digestion of human cells and amino acid specificity of TrypsINATOR. Comparison of a) TrypsINATOR-digested and b) TrypsINATOR LysCERATOR Mix-digested human cells to alternative products. Human cell lysates were digested with TrypsINATOR and three commercially available trypsin products (a), or TrypsINATOR LysCERATOR Mix and two alternative Trypsin/Lys-C mixtures (b) at enzyme-to-substrate ratios of 1:50 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/MS. Graphs show the number of unique peptides generated, grouped by the numbers of detected missed cleavages (a and b). c) Amino acid specificity of TrypsINATOR. Human cell lysate was digested using TrypsINATOR under the same conditions as described above, and the peptides were purified and analyzed correspondingly. The amino acid specificity was determined based on the frequency of the C-terminal amino acid from the identified peptides.

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