Lys-C (MS), CAS No.72561-05-8

CAS: 72561-05-8 Cat. No.: L1455941 EC Number: 276-716-4

GRADE & PURITY Animal Free, Carrier Free, Bioactive, Recombinant, Suitable for mass spectrometry (MS), ActiBioPure™, EnzymoPure™, for Protein sequencing, ≥70%(HPLC), ≥3 U/mg protein; expressed in E.coli

Accession #

Q7M135

Expression system

E. coli

Bioactivity

≥3 U/mg protein

Specifications

Product Name

Lys-C (MS), CAS No.72561-05-8

Synonyms

Lysyl Endopeptidase | Lysine Endonuclease | Lys-C

Grade

ActiBioPure™, Animal Free, Bioactive, Carrier Free, for Protein sequencing, Recombinant, Suitable for mass spectrometry (MS), EnzymoPure™

Specifications & Purity

Bioactive, Recombinant, suitable for mass spectrometry (MS), ActiBioPure™, EnzymoPure™, for protein sequencing, Animal Free, Carrier Free, ≥70%(HPLC), ≥3 U/mg protein; expressed in E.coli

Biochemical and Physiological Mechanisms

Highly specific endopeptidase that hydrolyzes lysyl bonds including the Lys-Pro bond. Preferential cleavage: Lys-|-Xaa, including Lys-|-Pro.

Bioactivity

≥3 U/mg protein

Accession #

Q7M135

Predicted molecular weight

27 kDa

CAS

72561-05-8

Enzyme Commission Number

3.4.21.50

Molecule Type

Enzyme

Product Line

Biological Products

Storage and Shipping

Concentration

≥3 U/mg protein; expressed in E.coli

Reconstitution

1mM HCl or 50 mM Acetic acid.

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Stability And Storage

Store at -20℃ (2 years). Upon reconstitution, it is recommended to aliquot. Avoid freeze/thaw cycle.

Unit definition

The amount of Lys-C enzyme that cleaves 1 µM Y-G-P Lys Nitroanilide per minute.

Application Protocols

Item-specific facts (from Product Data)

• Suitable for mass spectrometry (MS) and protein sequencing. No specific protocol steps, concentrations, or unit definitions are provided here; refer to CoA/Spec Sheet.

Representative workflow (general/literature; adapt to your sample)

1. Protein denaturation and reduction

• Solubilize protein in 6–8 M urea, 25–100 mM ammonium bicarbonate, pH ~8.0. Reduce disulfides with 5–10 mM DTT or 2–5 mM TCEP at 37°C for 30–60 min.

2. Alkylation

• Cool to RT; add IAA or CAA to 10–25 mM; incubate in dark 15–30 min. Quench excess alkylating agent.

3. Lys-C digestion

• Adjust urea to ≤8 M for Lys-C. Add Lys-C at E:S 1:50–1:100 (w/w). Incubate 2–4 h at 37°C for partial digest or overnight for exhaustive digest. Optionally dilute urea before adding trypsin for sequential digest.

4. Optional sequential trypsinization

• Dilute urea to ≤1–2 M. Add trypsin at E:S 1:50–1:100; incubate 4–16 h at 37°C.

5. Quench and cleanup

• Acidify to pH <3 with formic acid to stop digestion. Desalt using C18 StageTips/SPE. Dry and reconstitute in 0.1% formic acid for LC-MS.

Quality controls

• Include a standard protein (e.g., BSA or a known digest standard) to monitor specificity and recovery. Assess missed cleavages and peptide coverage by database search metrics.

Note

• The above is general guidance, not a specification for this item.

Biological Roles

General/literature context

• Origin: Lys-C (lysyl endopeptidase) is a bacterial serine protease, historically associated with Lysobacter species. Its physiological role in the native organism likely involves protein catabolism and nutrient acquisition.
• Enzymatic class: Serine endopeptidase; catalytic mechanism uses a Ser-His-Asp triad to hydrolyze peptide bonds after lysine residues.
• Substrate scope: Prefers peptide bonds where lysine is at the P1 position; activity can be modulated by protein folding, post-translational modifications, and proximal residues.
• Biochemical utility: In vitro, the enzyme is widely used for proteomics due to its strict specificity and robustness in denaturing conditions.

Item-specific note (from Product Data)

• This preparation is Recombinant, Animal Free, Carrier Free, and MS-suitable, aligning with analytical/biochemical applications rather than physiological studies.

Caveats

• No organism-specific biological activity claims are made for this product. Use is restricted to research applications as stated: For research use only.

Buffer Applications

Applicability

• Lys-C is an enzyme reagent, not a buffering compound. It is therefore not used to prepare buffer systems.

Practical notes (general/literature)

• Common digestion buffers: 25–100 mM ammonium bicarbonate (volatile, MS-friendly), or 50 mM Tris-HCl pH 8.0–8.5 for routine digests.
• Additives: Urea (up to ~8 M) facilitates denaturation; reduction (e.g., DTT/TCEP) and alkylation (IAA/CAA) improve accessibility before digestion.

For this item

• Specific buffer recommendations, excipients, and formulation are Not specified for this item; refer to CoA/Spec Sheet.

Green Alternatives

Context

• This product is a protein enzyme used for analytical proteolysis, not a solvent or commodity reagent. “Green alternatives” focuses on workflow choices that reduce environmental impact while maintaining MS-grade performance.

Greener workflow choices (general/literature)

• Buffer selection: Prefer volatile, low-toxicity buffers (e.g., ammonium bicarbonate) over non-volatile high-salt buffers to minimize solvent/salt waste and improve LC-MS compatibility.
• Denaturants: Use urea instead of guanidinium chloride when feasible; urea is less hazardous and easier to remove. Limit concentrations to the minimum that achieves solubilization.
• Surfactants: Choose MS-cleavable, biodegradable surfactants at minimal effective doses; avoid SDS to reduce downstream cleanup and wastewater burdens.
• Enzyme source: Recombinant, Animal Free supply (this item) reduces animal-derived inputs and associated environmental/ethical concerns.
• Miniaturization: Adopt micro- or nano-scale digestion (in-tip/on-bead) to decrease reagent volumes and waste.
• Solid-phase cleanup: Employ reusable SPE manifolds or StageTips with reduced plastic consumables; consolidate steps to limit single-use plastics.

Comparison table (general)

• Guanidinium chloride vs Urea: Higher solubilization power but more corrosive/toxic vs lower hazard and easier disposal.
• Non-volatile salts (e.g., phosphate) vs Ammonium bicarbonate: Strong buffering but non-volatile and LC-incompatible vs volatile and MS-friendly.

Note

• These are general sustainability suggestions; they are not specifications for this item.

Pharmaceutical Uses

Scope and applicability

• This product is supplied for research use only and is not an approved excipient or therapeutic. No clinical or medical uses are claimed or implied.

Relevant non-clinical/analytical roles (general)

• QC and characterization: Employed as a process analytical reagent for characterization of biotherapeutics (e.g., peptide mapping of monoclonal antibodies, evaluation of sequence variants and PTMs) under GMP-analogous analytical workflows.
• Sequencing support: Used in protein sequencing/mapping to generate defined peptide fragments that facilitate LC-MS/MS identification.

Item-specific notes (from Product Data)

• Recombinant, Animal Free, Carrier Free, MS-suitable grades align with stringent analytical testing where low background and high specificity are essential.

Documentation

• For any regulated use (e.g., in-process control or release testing), consult the CoA/Spec Sheet for available quality attributes and ensure appropriate qualification/validation under your quality system.

Physical Properties

Item-specific facts (from Product Data)

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight/formula: Not specified for this item; refer to CoA/Spec Sheet.

General/literature values for Lys-C (for context; not item specifications)

• Typical molecular mass (mature enzyme): ~30 kDa (literature; varies with source and tags).
• Isoelectric point (pI): often in the basic range (~9–10, literature), contributing to stability in alkaline buffers.
• Activity pH range: broad, ~7.5–10; optimal near pH 8.0–9.0 for many substrates (literature).
• Temperature profile: active at 25–37°C; denatures at elevated temperatures typical of proteins (literature).
• Denaturant tolerance: retains activity in high urea (up to ~8 M) and moderate guanidinium chloride concentrations (literature), which is advantageous for proteomics sample prep.
• Solubility: water-buffer soluble; avoid organic co-solvents that denature or inactivate serine proteases (literature).

Practical implications

• Because MS-grade workflows often require chaotropes and surfactants, Lys-C’s denaturant tolerance (literature) is leveraged for digestion of difficult, insoluble, or membrane-rich samples.
• Do not treat any numerical values above as specifications for this catalog item; for lot-specific attributes (concentration of supplied solution, excipients, stabilizers, or lyophilized mass), consult the CoA/spec sheet.

Quality & Grades

Item-specific facts (from Product Data)

• Grades/attributes: Animal Free; Carrier Free; Bioactive; Recombinant; Suitable for mass spectrometry (MS); ActiBioPure™; EnzymoPure™; for Protein sequencing.

Interpretation and implications

• Recombinant, Animal Free: Produced without animal-derived raw materials, reducing adventitious agent and endotoxin concerns and improving regulatory acceptance for analytical use.
• Carrier Free: No protein carriers (e.g., BSA, gelatin) are added—critical to avoid background peptides/proteins in proteomics workflows and to ensure accurate peptide mass mapping.
• Bioactive: Functionally active protease, validated by activity assay (assay details not specified for this item; refer to CoA/Spec Sheet).
• Suitable for MS: Manufactured and handled to minimize contaminants (e.g., keratins, polyethylene glycols, polymeric surfactants, salts) that interfere with LC-MS detection. Low autolysis background is typically targeted in MS-grade enzymes (general note).
• ActiBioPure™ / EnzymoPure™: Proprietary quality designations indicating high purity and performance; consult product documentation for exact acceptance criteria.
• For Protein sequencing: Indicates utility in Edman/peptide mapping workflows, where clean and specific cleavage is required.

What to check on the CoA/spec sheet

• Specific activity (units/mg), residual host-cell proteins/DNA, endotoxin level, protease contaminants, formulation (lyophilized vs solution), stabilizers, and recommended working concentration. If not listed here, these are Not specified for this item; refer to CoA/Spec Sheet.

Reaction & Applications

Item-specific focus (from Product Data)

• Intended use: Suitable for mass spectrometry (MS); for protein sequencing. Carrier Free and Recombinant design support high-fidelity proteolysis for analytical workflows.

General/literature applications of Lys-C

• Proteomics sample preparation: Site-specific cleavage C-terminal to lysine residues generates peptide pools tailored for LC-MS/MS. Often used alone or in tandem with trypsin (sequential Lys-C → Trypsin) to improve sequence coverage, especially for membrane proteins and basic regions.
• Limited proteolysis and domain mapping: Controlled digestion to probe protein folding, accessibility, and domain boundaries.
• Disulfide mapping: After reduction/alkylation, Lys-C facilitates mapping of cysteine-containing peptides with higher confidence.
• Top-down/bottom-up strategies: Bottom-up workflows rely on predictable Lys-C specificity. In top-down MS, Lys-C may be used to simplify very large proteins into megadalton-scale subunits (limited proteolysis), though less common.
• Glycoproteomics: Pre-digestion with Lys-C can improve subsequent trypsin performance on glycoproteins by reducing structural constraints (literature).

Practical notes (literature)

• Specificity: Cleaves on the C-terminal side of Lys residues; cleavage is typically reduced at Lys–Pro sites.
• Denaturant tolerance: Active in up to ~8 M urea; consider diluting before adding trypsin in sequential protocols.
• Ratios/kinetics: Typical enzyme:substrate (E:S) 1:50 to 1:200 (w/w), 1–4 h at 37°C for partial digests or overnight for exhaustive digests, depending on matrix and denaturants.
• Peptide cleanup: Use StageTips or SPE cartridges before LC-MS to remove salts/denaturants.

Reaction Conditions

Item-specific facts (from Product Data)

• Storage: -20°C; avoid repeated freeze–thaw.
• No item-specific working conditions are specified; refer to CoA/Spec Sheet.

General/literature digestion conditions for Lys-C (guidance only)

• Buffer: 25–100 mM ammonium bicarbonate or 50 mM Tris-HCl, pH 8.0–9.0.
• Temperature: 25–37°C; 37°C is common for efficient digestion.
• Enzyme:substrate (E:S): 1:50 to 1:200 (w/w) for overnight exhaustive digestion; 1:20 to 1:100 for shorter/limited digests.
• Denaturants: Up to ~8 M urea compatible; for sequential Lys-C → Trypsin, dilute urea to ≤1–2 M before adding trypsin (literature).
• Reduction/alkylation: Reduce with 5–10 mM DTT or 2–5 mM TCEP (30–60 min, 37°C), alkylate with 10–25 mM IAA or CAA (in dark, RT, 15–30 min). Quench excess alkylating agent prior to digestion.
• Duration: 1–4 h for partial digestion; 8–16 h for exhaustive digestion, depending on sample complexity and accessibility.
• Inhibitors: Avoid serine protease inhibitors (e.g., PMSF, AEBSF) and heavy-metal contaminants.

Expected outcomes (literature)

• High specificity for C-terminal Lys cleavage; reduced cleavage at Lys–Pro motifs. Resulting peptides are well-suited for LC-MS/MS, often improving sequence coverage of basic and membrane proteins.

Safety & Handling

Item-specific facts (from Product Data)

• GHS classification, pictograms, signal word, H-statements: Not specified for this item; refer to SDS.
• Storage conditions: Store at -20°C. Avoid repeated freezing and thawing.
• Shipped in: Ice chest + Ice pads.

General safety guidance for proteolytic enzymes (literature/standard practice)

• Hazards: Proteases can be respiratory/skin sensitizers. Avoid aerosolization and inhalation; minimize dust formation if lyophilized. Handle solutions with care to avoid accidental exposure to eyes/skin.
• PPE: Lab coat, nitrile gloves, and safety goggles. Use a fume hood or biosafety cabinet when preparing stocks to limit aerosols.
• Incompatibilities: Strong oxidizers and heavy-metal ions can inactivate serine proteases; extreme pH (<5 or >11) and organic solvents can denature proteins (literature).
• First aid (overview; defer to SDS): In case of skin/eye contact, rinse with plenty of water; if inhaled, move to fresh air; if ingested, rinse mouth with water. Seek medical advice if symptoms persist.
• Spill response: Avoid raising dust/aerosols. Gently dampen lyophilized spills before collection. Dispose according to institutional and local regulations.
• Waste: Treat as laboratory biological reagent waste; do not discharge active enzyme to drains without deactivation (e.g., heat/denaturant) unless permitted by local rules.

Always consult the product’s SDS for authoritative hazard classification and response procedures.

Solvent Selection

Applicability

• As a protein enzyme reagent, “solvent” selection is effectively buffer selection. Organic solvents used for small-molecule chemistry are not applicable.

General/literature guidance for Lys-C buffers

• Preferred buffers: Tris-HCl or HEPES, pH 8.0–9.0 (literature). Ammonium bicarbonate (NH4HCO3, 25–100 mM) is popular for LC-MS sample prep due to volatility.
• Salt/volatility: For MS workflows, use volatile buffers (e.g., ammonium bicarbonate) during digestion to simplify downstream desalting.
• Denaturants: Lys-C retains activity in high urea (commonly up to 8 M) and tolerates moderate guanidinium chloride, enabling digestion of difficult proteins (literature). Reduce denaturant concentration prior to adding trypsin in sequential digestions.
• Detergents: Some MS-compatible surfactants (e.g., acid-cleavable types) can be used; conventional SDS inhibits MS and can inhibit enzymes unless greatly diluted. Validate compatibility empirically.
• Reducing/alkylating agents: DTT or TCEP reduction followed by iodoacetamide (IAA) or chloroacetamide (CAA) alkylation enhances accessibility; ensure reagents are quenched/controlled to avoid overalkylation (literature).

Comparison (general)

• Lys-C vs Trypsin: Lys-C cleaves after Lys only (Trypsin cleaves after Lys and Arg). Lys-C shows superior tolerance to chaotropes, making it preferable for initial digests of highly denatured samples.
• Lys-C vs Lys-N: Lys-N cleaves N-terminal to Lys and yields different peptide ladders; selection depends on mapping strategy and MS method.

Storage & Reconstitution

Item-specific facts (from Product Data)

• Storage conditions: Store at -20°C. Avoid repeated freezing and thawing.
• Shipping: Ice chest + Ice pads.
• Reconstitution details, concentration, buffer formulation, stabilizers: Not specified for this item; refer to CoA/Spec Sheet.

General/literature guidance

• If lyophilized: Reconstitute in MS-compatible buffer or mild acid (e.g., 10–50 mM acetic acid) to a convenient stock concentration. Allow to dissolve gently on ice; avoid vigorous vortexing to limit foaming.
• If supplied in solution: Aliquot immediately upon receipt to single-use volumes to prevent freeze–thaw cycles. Snap-freeze aliquots (e.g., in a dry ice/ethanol bath) and store at -20°C or below.
• Working buffer: Dilute stocks into digestion buffer immediately before use. Maintain on ice during setup; warm to reaction temperature only after mixing with substrate.
• Stability considerations: Avoid protease inhibitors and metal contaminants. For extended storage, -80°C may improve longevity (literature); verify compatibility with the specific formulation.
• Thawing: Thaw on ice. Mix by gentle inversion; brief, low-speed spin-down can collect condensate.

Documentation

• For exact reconstitution solvent, target concentration, and on-label stability/expiration for this SKU, consult the CoA/Spec Sheet. For research use only.

Structure & Identity

Item-specific facts (from Product Data)

• Product name: Lys-C (MS); CAS: 72561-05-8
• Grade/Purity: Animal Free, Carrier Free, Bioactive, Recombinant, Suitable for mass spectrometry (MS), ActiBioPure™, EnzymoPure™, for Protein sequencing
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES: Not applicable to proteins.
• InChIKey: Not applicable to proteins.

General/literature description

• Identity: Endoproteinase Lys-C (lysyl endopeptidase), typically derived from Lysobacter species (literature). It is a serine protease with strict preference for cleaving peptide bonds on the C-terminal side of lysine residues (K) in proteins/peptides.
• Structural features (literature): Single-chain protease (typical mature enzyme ~30 kDa), catalytic Ser-His-Asp triad common to chymotrypsin-like serine proteases. No small-molecule line notation applies.
• 2D/sequence-level description (literature): Polypeptide enzyme with an active-site serine positioned in a β-barrel fold; substrate-binding pocket favors ε-amino group environment of lysine at P1.

Notes

• Recombinant and Animal-Free indicates production without animal-derived materials; Carrier Free denotes no protein carriers (e.g., BSA) are added—important for MS workflows.
• For exact accession, sequence, and calculated mass of this specific lot, consult the product CoA/specification sheet.

Synthetic Utility

Applicability

• Traditional “synthetic utility” for small-molecule transformations does not apply. However, in protein/peptide chemistry, Lys-C is a precise biocatalyst enabling controlled bond cleavage.

General/literature utility in protein chemistry

• Site-specific proteolysis: Generates peptides terminating in lysine (at the C-terminus), simplifying database search parameters for bottom-up proteomics.
• Orthogonal digestion strategies: Combined with trypsin, Glu-C, Asp-N, or Lys-N to modulate peptide length and coverage; sequential Lys-C → Trypsin often enhances proteome coverage and quantification robustness.
• Limited proteolysis mapping: Probes structural dynamics and domain accessibility; useful in epitope exposure studies and stability assessments.
• PTM analysis: Facilitates mapping of phosphorylation, glycosylation, deamidation, and oxidation when paired with appropriate enrichment workflows and MS methods.

Item-specific note (from Product Data)

• The Carrier Free, MS-suitable, Recombinant preparation supports low-background peptide maps, essential for high-sensitivity LC-MS/MS and protein sequencing experiments.

Target Specificity

Applicability

• This section typically applies to antibodies or affinity reagents. Lys-C is a proteolytic enzyme, not an affinity reagent.

General/literature specificity of Lys-C (for context)

• Cleavage preference: Carboxyl side of lysine residues (K) in polypeptides; reduced activity at Lys–Pro sites.

Item-specific note

• No antigen/epitope/clone/isotype information applies. Target specificity in the immunological sense is Not applicable.