Science & Applications

Understanding LysN mechanism and applications in proteomics research

How LysN Works

N-terminal lysine cleavage for complementary proteomics coverage

LysN Cleavage

...Peptide-↓K-Peptide...

Cleaves on the N-terminal side of lysine residues, generating peptides with C-terminal lysine.

Trypsin Cleavage

...Peptide-K↓-Peptide...

Cleaves on the C-terminal side of lysine/arginine, generating peptides with N-terminal K/R.

Why This Matters

The opposite cleavage specificity generates completely different peptide sets:

  • Complementary Coverage: Fills gaps in protein sequence coverage missed by trypsin
  • Different Charge States: C-terminal lysine vs N-terminal lysine affects peptide ionization
  • Improved Ion Mobility: LysN peptides show greater spread in the ion mobility dimension
  • Alternative PTM Sites: Access to post-translational modifications in different peptide contexts

Key Applications

Aβ Biomarker Assays

N- and C-terminal Measurement: Lys-N digestion overcomes challenges of measuring terminal regions of Aβ.

Clinical Utility: Essential for sensitive LC-MS/MS quantification of Aβ40 and Aβ42 in CSF and blood biomarker assays.

Data-Independent Acquisition (DIA)

Enhanced Separation: Greater spread in ion mobility dimension.

Better Identification: Improved confidence in complex samples.

Protein Characterization

Orthogonal Digestion: Complementary peptide sets for improved coverage.

PTM Mapping: Access modifications in different contexts.

Quantitative Proteomics

Label-Free & Multiplexed: Compatible with TMT, iTRAQ, and SILAC.

Reproducible: Consistent cleavage efficiency.

Frequently Asked Questions

LysN cleaves on the N-terminal side of lysine (↓K) while trypsin cleaves on the C-terminal side of lysine and arginine (K/R↓). This generates completely different peptide sets with complementary sequence coverage.

LysN doesn't express well in recombinant systems, requiring purification from natural sources (mushrooms). This makes production more challenging than recombinant enzymes like trypsin. We've developed proprietary purification methods to ensure consistent supply.

Yes, LysN can be used as an alternative to trypsin for many proteomics applications. It's particularly valuable for DIA proteomics and when you need different peptide coverage. Many researchers use both enzymes for comprehensive protein characterization.

LysN-generated peptides have greater spread in the ion mobility dimension compared to tryptic peptides. This reduces peak overlap and co-elution, improving peptide identification confidence in complex samples.

We recommend 1:50 to 1:100 (w/w) enzyme:substrate ratio. Start with 1:50 for difficult proteins and 1:100 for routine digestions. Overnight incubation (16 hours) at 37°C typically provides complete digestion.

Compatible with isobaric and metabolic labeling strategies (e.g., TMT®, iTRAQ®, Stable isotope labeling of cells in culture (SILAC)).

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