# Lysyl endopeptidase
**Endoproteinase Lys-C** (Lys-C, lysyl endopeptidase) is a lysine-specific serine endoprotease secreted by the Gram-negative soil bacterium *Lysobacter enzymogenes* that hydrolyzes peptide bonds on the carboxyl side of lysine residues, including the Lys-Pro bond. It is classified as EC 3.4.21.50 and is sold under the name Endoproteinase Lys-C by Roche and other suppliers for protein sequencing and proteomics sample preparation.[1](https://www.uniprot.org/uniprotkb/Q7M135/entry) • [2](https://www.merckmillipore.com/deepweb/assets/sigmaaldrich/product/documents/217/458/p3428bul.pdf)
| Key fact | Detail |
|---|---|
| Source and class | Serine endoprotease from *Lysobacter enzymogenes*; EC 3.4.21.50; peptidase family S1 (trypsin-like)[1](https://www.uniprot.org/uniprotkb/Q7M135/entry) • [3](https://www.rcsb.org/annotations/4NSY) |
| Cleavage rule | Preferential cleavage Lys-\|-Xaa, including Lys-\|-Pro; Lys-Glu bonds are also cleaved, with some minor non-specific cleavage reported[1](https://www.uniprot.org/uniprotkb/Q7M135/entry) • [2](https://www.merckmillipore.com/deepweb/assets/sigmaaldrich/product/documents/217/458/p3428bul.pdf) |
| Denaturant tolerance | Remains active in 8 M urea, 2 M guanidine-HCl, 1% SDS, 2% CHAPS, and 40% acetonitrile; maximal activity at pH 8[4](https://www.thermofisher.com/order/catalog/product/90057) |
| Molecular mass | About 30 kDa (lyophilized MS-grade enzyme); 33 kDa by reducing SDS-PAGE for the Roche sequencing-grade product[4](https://www.thermofisher.com/order/catalog/product/90057) • [5](https://www.merckmillipore.com/IN/en/product/roche/endolyssro) |
| Typical use | Sequential Lys-C then trypsin digestion in bottom-up proteomics; in a 293T cell study this raised identified proteins from 2,070 to 2,317 (an 11.9% increase)[6](https://www.sciencedirect.com/science/article/pii/S1872204017609988) |
| Working conditions | Enzyme-to-protein ratios of 1:20 to 1:100 (w/w); incubation 2 to 18 hours at 37 °C[2](https://www.merckmillipore.com/deepweb/assets/sigmaaldrich/product/documents/217/458/p3428bul.pdf) |
| Inhibitors | Aprotinin, DFP, leupeptin, and TLCK[7](https://www.merckmillipore.com/SG/en/product/mm/324715) |
## Enzymology
Lys-C is a trypsin-type serine protease with a high pH optimum that hydrolyzes Lys-Xaa peptide bonds.[8](https://pubmed.ncbi.nlm.nih.gov/25004961/) In ammonium bicarbonate or Tris-HCl buffers at pH 8.5 it cleaves specifically at the carboxyl side of lysine, producing a limited number of fragments, which is the property that made it useful for protein sequencing work.[2](https://www.merckmillipore.com/deepweb/assets/sigmaaldrich/product/documents/217/458/p3428bul.pdf) The Roche sequencing-grade preparation has an optimum pH of 8.5 to 8.8 and a specific activity of at least 200 units/mg protein.[5](https://www.merckmillipore.com/IN/en/product/roche/endolyssro) Unlike trypsin, Lys-C cleaves lysines followed by prolines, and its prototypic peptides typically carry higher charge states.[4](https://www.thermofisher.com/order/catalog/product/90057)
Its most distinctive practical property is stability under strongly denaturing conditions. Lys-C retains most of its activity in 2.0 M urea or 0.1% SDS, and MS-grade preparations remain active in 8 M urea, 2 M guanidine-HCl, 1% SDS, 2% CHAPS, and 40% acetonitrile.[2](https://www.merckmillipore.com/deepweb/assets/sigmaaldrich/product/documents/217/458/p3428bul.pdf) • [4](https://www.thermofisher.com/order/catalog/product/90057) This allows digestion directly on denatured, unfolded protein without prior desalting or buffer exchange, which is why Lys-C is often the first enzyme applied in a proteomics workflow.[9](https://www.mdpi.com/1424-8247/16/9/1327)
## Structure
The crystal structures of wild-type Lys-C and its K30R variant, covalently inhibited by N(α)-p-tosyl-lysyl chloromethylketone (TLCK), were determined to 1.1 and 0.9 Å resolution (PDB entries 4NSV and 4NSY), giving unusually precise views of the active and substrate-binding sites for this enzyme subfamily.[8](https://pubmed.ncbi.nlm.nih.gov/25004961/) • [3](https://www.rcsb.org/annotations/4NSY) The structures show a trypsin-like fold with a catalytic triad at His57, Asp113, and Ser194 (LepA numbering for the homologous lysyl endopeptidase), and lysine specificity conferred by Asp225.[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC451660/) The high pH optimum is attributed to electrostatic effects of an aromatic Tyr/His stack on the catalytic aspartate, a feature shared across this enzyme subfamily. Unexpectedly, the electron density indicates that a hydroxyl group, rather than the expected oxyanion, binds in the partially solvent-exposed oxyanion hole, suggesting that protonation of the alkoxide catalytic intermediate may recur during serine protease catalysis.[8](https://pubmed.ncbi.nlm.nih.gov/25004961/)
## History
The enzyme's value for primary structure determination was described by Peter A. Jekel, Wicher J. Weijer, and Jaap J. Beintema of the [University of Groningen](https://www.edgepedia.org/university-of-groningen) in *Analytical Biochemistry* in 1983, in a paper titled "Use of endoproteinase Lys-C from *Lysobacter enzymogenes* in protein sequence analysis"; the enzyme was already commercially available at that time.[11](https://www.sciencedirect.com/science/article/pii/0003269783903081) *Lysobacter enzymogenes* also secretes related proteases, including α-lytic protease, a chymotrypsin-like bacterial serine protease, and an arginyl endopeptidase (endoproteinase Arg-C) whose gene was cloned in 1998.[12](https://pubmed.ncbi.nlm.nih.gov/9878833/)
## Use in proteomics
In bottom-up mass spectrometry proteomics, proteins are digested into peptides for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Trypsin is the standard protease because it generates peptides with basic C-terminal residues, but it cleaves less efficiently after lysine than after arginine, particularly when the lysine is followed by proline or flanked by acidic residues, producing missed cleavages.[6](https://www.sciencedirect.com/science/article/pii/S1872204017609988) • [4](https://www.thermofisher.com/order/catalog/product/90057)
**Sequential digestion.** The common remedy is Lys-C/trypsin sequential digestion: the protein is first digested with Lys-C while still dissolved in high urea, then the urea is diluted and trypsin is added. In the protocol used by Qian and colleagues on a 293T human renal epithelial cell extract, the initial Lys-C digest ran 3 hours at a 1:100 enzyme-to-protein ratio (w:w) in 8 M urea, after which the sample was diluted to 2 M urea for a 16-hour trypsin digest at 1:50, all in 100 mM ammonium bicarbonate, pH 8.0, at 37 °C.[6](https://www.sciencedirect.com/science/article/pii/S1872204017609988) The tandem digest identified 2,317 proteins against 2,070 for trypsin alone, an 11.9% increase, and reduced missed cleavage sites, especially at lysine (K) sites, while raising average sequence coverage.[6](https://www.sciencedirect.com/science/article/pii/S1872204017609988) Commercial trypsin/Lys-C mixes are sold for the same purpose, since trypsin alone does not fully digest proteins at the carboxyl end of lysine and arginine residues.[4](https://www.thermofisher.com/order/catalog/product/90057)
**Other applications.** Because Lys-C produces longer peptides than trypsin, it serves as an alternative protease for increased peptide length, multiple-protease digestion strategies, and oxygen-18 labeling.[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC3751594/) In biotherapeutics, Lys-C peptide mapping is used for sequence confirmation and structural characterization of antibodies, including disulfide bond mapping; digestion under neutral pH with methionine as a scavenger minimizes deamidation and oxidation artifacts, and optimized methods have achieved 100% sequence coverage for a monoclonal antibody.[14](https://www.aladdinsci.com/us_en/faqs/endoproteinase-lys-c-mechanism-applications-and-method-control-en.html) • [9](https://www.mdpi.com/1424-8247/16/9/1327) Lys-C digestion is also used in phosphopeptide enrichment workflows because it generates peptides with primary amines at both termini, allowing double labeling with amine-reactive isobaric tags.[4](https://www.thermofisher.com/order/catalog/product/90057)
## Related enzymes and variants
Lysine-specific peptidases related to Lys-C occur in other bacteria: lysyl endopeptidases have been found in the culture broths of *Achromobacter lyticus*, *Lysobacter enzymogenes*, and *Pseudomonas aeruginosa*, and a second lysine-specific serine protease (LepB) was identified in *[Lysobacter](https://www.edgepedia.org/lysobacter)* sp. strain IB-9374.[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC451660/) Peptidyl-Lys metalloendopeptidases (PKMs), which cleave at lysine residues in the P1 position, function as complementary enzymes to Lys-C and can be combined with it to distinguish protein N- and C-termini in a single experiment.[15](https://link.springer.com/content/pdf/10.1007/s00253-025-13473-7.pdf) On the arginine side, a combined Lys-C/Arg-C Ultra protocol for data-independent acquisition proteomics achieved more than 99% arginine cleavage efficiency and 95% lysine cleavage efficiency, cutting arginine missed cleavages from about 30% to under 1% relative to a standard Lys-C/trypsin protocol.[16](https://pmc.ncbi.nlm.nih.gov/articles/PMC12338676/)
## Handling
The lyophilized sequencing-grade enzyme is stored desiccated at 2 to 8 °C and reconstituted in water; after reconstitution, aliquots can be frozen for several weeks, and stock solutions are stable for up to 2 days at 4 °C or up to 1 month at −20 °C.[2](https://www.merckmillipore.com/deepweb/assets/sigmaaldrich/product/documents/217/458/p3428bul.pdf) • [7](https://www.merckmillipore.com/SG/en/product/mm/324715) Self-digestion may occur at temperatures above 37 °C, and a standard peptide such as melittin or oxidized insulin B chain is recommended as a control substrate.[2](https://www.merckmillipore.com/deepweb/assets/sigmaaldrich/product/documents/217/458/p3428bul.pdf)
## References
1. [Lysyl endopeptidase (Q7M135, LYSC_LYSEN), UniProtKB](https://www.uniprot.org/uniprotkb/Q7M135/entry)
2. [Endoproteinase Lys-C (P3428) Technical Bulletin, Sigma-Aldrich](https://www.merckmillipore.com/deepweb/assets/sigmaaldrich/product/documents/217/458/p3428bul.pdf)
3. [Annotations: 4NSY, RCSB Protein Data Bank](https://www.rcsb.org/annotations/4NSY)
4. [Mass Spectrometry Grade Proteases, Thermo Scientific product 90057](https://www.thermofisher.com/order/catalog/product/90057)
5. [Endoproteinase Lys-C Sequencing Grade from Lysobacter enzymogenes, Roche/Sigma-Aldrich](https://www.merckmillipore.com/IN/en/product/roche/endolyssro)
6. [Qian et al. (2017). Evaluation of Endoproteinase Lys-C/Trypsin Sequential Digestion Used in Proteomics Sample Preparation. Chinese Journal of Analytical Chemistry 45(3):316-321](https://www.sciencedirect.com/science/article/pii/S1872204017609988)
7. [Endoproteinase Lys-C, Sequencing Grade, product 324715, Sigma-Aldrich](https://www.merckmillipore.com/SG/en/product/mm/324715)
8. [Asztalos et al. (2014). Atomic resolution structure of a lysine-specific endoproteinase from Lysobacter enzymogenes suggests a hydroxyl group bound to the oxyanion hole. Acta Crystallographica Section D 70:1832-1843](https://pubmed.ncbi.nlm.nih.gov/25004961/)
9. [Expanding the Analytical Toolbox: Developing New Lys-C Peptide Mapping Methods with Minimized Assay-Induced Artifacts to Fully Characterize Antibodies. Pharmaceutics 16(9):1327 (2023)](https://www.mdpi.com/1424-8247/16/9/1327)
10. [Ohnishi et al. (2003). A Second Lysine-Specific Serine Protease from Lysobacter sp. Strain IB-9374. Journal of Bacteriology](https://pmc.ncbi.nlm.nih.gov/articles/PMC451660/)
11. [Jekel, Weijer, Beintema (1983). Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis. Analytical Biochemistry 134(2):347-354](https://www.sciencedirect.com/science/article/pii/0003269783903081)
12. [Wright, Graham, Jennings (1998). Cloning of a Lysobacter enzymogenes gene that encodes an arginyl endopeptidase (endoproteinase Arg-C). Biochimica et Biophysica Acta 1443(3):369-374](https://pubmed.ncbi.nlm.nih.gov/9878833/)
13. [Protein Analysis by Shotgun/Bottom-up Proteomics, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC3751594/)
14. [Endoproteinase Lys-C: Mechanism, Applications, and Method-Control Considerations for Lysine-Specific Digestion, Aladdin Scientific](https://www.aladdinsci.com/us_en/faqs/endoproteinase-lys-c-mechanism-applications-and-method-control-en.html)
15. [Production and application of peptidyl-Lys metalloendopeptidase: advances, challenges, and future perspectives. Applied Microbiology and Biotechnology (2025)](https://link.springer.com/content/pdf/10.1007/s00253-025-13473-7.pdf)
16. [Synergistic Arg-C Ultra and Lys-C Digestion for Quantitative Proteomics, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC12338676/)
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*Topic: Encyclopedia › Life and health › Biological foundations › Biochemistry and metabolism › Enzyme classes and activities › Proteolytic and peptidase enzymes › Applied and research proteases › Proteases as laboratory reagents*
*Initially written Sep 17, 2026 · Reviewed: Oct 21, 2026 · Edited: Oct 11, 2026; Oct 15, 2026; Oct 21, 2026 · Last review: Oct 21, 2026*
*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*
License: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license