Lys-C (MS), CAS No.72561-05-8‌

CAS: 72561-05-8‌ Cat. No.: L1523246

GRADE & PURITY Recombinant, Suitable for mass spectrometry (MS), EnzymoPure™, ≥99%(HPLC), ≥25 U/mg protein

Expression system

E. coli

Overview

MS-grade Lys-C protease specifically cleaves peptide bonds at the C-terminus of lysine residues. It can improve the digestion of hard-to-hydrolyze proteins and is recommended for use in 6–8 M urea solutions. Reconstitution Solution: Reconstitute Lys-C protease in 50 mM acetic acid buffer. pH Range: rLys-C exhibits optimal activity at pH 8–9. Application: Lys-C protease specifically cleaves lysine (K) residues in denatured proteins that are resistant to proteolysis. It is recommended for use in combination with trypsin. Recommended SOP for Mixed Protease Digestion: A. General In-Solution Digestion Use a mixture of 100 μg trypsin and 20 μg Lys-C at a protein-to-protease ratio (w/w) of 25:1. Incubate overnight at 37°C for 13–16 hours. Tip: Urea concentration should be <1 M; use 50 mM Tris-HCl buffer (pH 8). B. 1+1 Step In-Solution Digestion 1. Use a mixture of 100 μg trypsin and 20 μg Lys-C at a protein-to-protease ratio (w/w) of 25:1. Incubate at 37°C for 2–4 hours. Tip: Urea concentration should be 6–8 M; use 50 mM Tris-HCl buffer (pH 8). 2. Dilute the solution 6-fold and incubate overnight at 37°C for 13–16 hours. Tip: Urea concentration should be <1 M; use 50 mM Tris-HCl buffer (pH 8).

Specifications

Product Name

Lys-C (MS), CAS No.72561-05-8‌

Grade

Recombinant, Suitable for mass spectrometry (MS), EnzymoPure™

Specifications & Purity

EnzymoPure™,suitable for mass spectrometry (MS),Recombinant,≥99%(HPLC),≥25 U/mg protein

CAS

72561-05-8‌

Enzyme Commission Number

EC 3.4.21.50

Molecule Type

Enzyme

Product Line

biological_products

Storage and Shipping

Concentration

≥25 U/mg protein

Storage

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

Shipped In

Ice chest + Ice pads

Unit definition

The amount of rLys C that hydrolyzes 1.0 μmol of T-G-P Lys Nitroanilid per minute at 405 nm, under conditions of pH 7.8 and 25°C.

Application Protocols

The following protocols are general, literature-based workflows for MS sample preparation with Lys-C. They are provided for guidance and are not item-specific instructions.

A. Standard denaturing Lys-C digest (bottom-up proteomics)

1. Solubilize protein at 1–5 mg/mL in 50 mM ammonium bicarbonate with 6–8 M urea.
2. Reduce (e.g., 5–10 mM DTT, 45 min, 56–60°C) and cool to RT; alkylate (e.g., 10–25 mM IAA, 20–30 min, dark, RT). Quench excess IAA (e.g., add equimolar DTT).
3. Add Lys-C at 1:50–1:100 (w/w, enzyme:protein). Incubate 2–4 h at 37°C.
4. For sequential digestion, dilute urea to ≤1–2 M and add trypsin (1:50–1:100) for an additional 2–12 h at 37°C.
5. Desalt peptides (e.g., C18 SPE), dry, and reconstitute in 0.1% formic acid for LC–MS/MS.

B. Limited proteolysis (mapping domains)

1. Buffer exchange protein into 25–50 mM Tris-HCl, pH 8.0.
2. Add Lys-C at low ratios (1:200–1:1000). Incubate on ice to 25°C for 5–60 min.
3. Quench (e.g., rapid acidification to 0.5–1% FA) and analyze fragments by MS or SDS-PAGE.

Notes:

• Avoid excessive SDS; consider MS-cleavable surfactants at ≤0.1% if needed.
• Use LC–MS-grade reagents and clean plasticware to minimize contamination.

Item-specific reconstitution buffer, concentration, and unit definition are Not specified for this item; refer to CoA/Spec Sheet.

Biological Roles

Item-specific biological role claims are not provided; this reagent is sold strictly for research use.

General/literature context for Lys-C:

• Origin: Lysyl endopeptidase activity was originally characterized from Lysobacter enzymogenes. Recombinant forms are widely used as research tools.
• Enzymatic function: Serine protease that catalyzes hydrolysis of peptide bonds after lysine residues. In native microbial contexts, such proteases contribute to nutrient acquisition and protein turnover.
• Research utility in biology/biochemistry: Enables controlled proteolysis to interrogate protein structure, domain organization, and post-translational modifications. In proteomics, Lys-C-generated peptides complement tryptic peptides, enhancing sequence coverage, PTM localization, and quantitation fidelity.
• Compatibility with denaturants: Activity in urea facilitates digestion of structurally resilient protein complexes, inclusion bodies, or membrane proteins under denaturing conditions (literature), improving peptide accessibility and reducing missed cleavages.

Note: No physiological/clinical claims are made for this product. Any biological pathway descriptions above are general background rather than item-specific properties. For applications in living systems or diagnostics, follow institutional approvals and do not use outside research workflows.

Buffer Applications

Because Lys-C is an enzyme rather than a buffering compound, it is not used to establish buffer pH per se. Instead, successful use depends on selecting compatible digestion buffers.

General, literature-based buffer guidance (not item-specific):

• MS-friendly choice: 25–100 mM ammonium bicarbonate, pH 8.0–8.5. Volatile; simplifies post-digest cleanup and LC–MS.
• Robust buffering (non-volatile): 50 mM Tris-HCl, pH 8.0–8.5, or 50 mM HEPES, pH 8.0. Use desalting prior to MS to remove non-volatiles.
• Denaturing workflows: 4–8 M urea in the chosen buffer can be employed to unfold proteins while maintaining Lys-C activity (literature). Gradual dilution may be applied before secondary digestion (e.g., trypsin).
• Additives: Reduction (e.g., 5–10 mM DTT or 2–5 mM TCEP) and alkylation (e.g., 10–25 mM IAA/CAA) are commonly integrated. Avoid high concentrations of SDS; consider MS-cleavable surfactants at 0.01–0.1% if necessary.

Practical notes:

• Maintain pH near 8–9 for optimal activity (literature). Extreme pH or non-volatile salts can inhibit the enzyme and complicate MS.
• Filter buffers (0.22 µm) and use LC–MS-grade reagents to minimize background.

Item-specific formulation, stabilizers, and recommended buffers are Not specified for this item; refer to CoA/Spec Sheet.

Green Alternatives

Lys-C is itself a biocatalyst and often represents a greener alternative to harsher chemical cleavage methods.

Comparison (general, literature):

• Approach: Enzymatic cleavage (Lys-C)

  • Advantages: Aqueous media, mild temperatures (~37°C), high site-selectivity (after Lys), low hazardous waste, compatible with MS workflows.
  • Trade-offs: Proteinaceous reagent (storage at −20°C), sensitive to extreme pH/solvents; requires careful buffer selection.

• Chemical cleavage: CNBr (cleaves after Met) in strong acid

  • Advantages: No enzyme; single-step chemistry.
  • Trade-offs: Uses toxic CNBr and strong acids (e.g., formic acid), generates hazardous waste, less control over side reactions and sample integrity; peptides may be less MS-friendly without additional cleanup.

• Trypsin vs. Lys-C

  • Trypsin: Extremely common, cleaves after Lys/Arg, but loses activity in high urea and can be inhibited by detergents.
  • Lys-C: Superior stability in urea (up to ~8 M literature), useful for denaturing workflows and sequential digests.

Green practice tips (general):

• Choose volatile buffers (ammonium bicarbonate) to minimize non-volatile waste and simplify post-digest cleanup.
• Use minimal surfactant amounts and MS-cleavable surfactants when necessary.
• Optimize enzyme:substrate ratios and digestion times to reduce reagent consumption and reprocessing.

Pharmaceutical Uses

No excipient or therapeutic claims are made for this product. It is intended for laboratory research use only.

General/industry practice (non-clinical, not item-specific):

• Process analytics and QC: Lys-C is widely used in biopharmaceutical development and quality control for peptide mapping of therapeutic proteins (e.g., antibodies, fusion proteins) to confirm sequence, monitor post-translational modifications (deamidation, oxidation, glycation), and assess product consistency.
• Identity testing: Enzymatic peptide mapping with Lys-C provides orthogonal confirmation of primary structure relative to tryptic maps, improving confidence in identity and coverage of Lys-rich regions.
• Stability studies: Controlled digests can reveal degradation hotspots and facilitate forced-degradation assessments.
• Comparability: Used alongside other proteases (trypsin, Glu-C, Asp-N) to provide complementary maps during process changes or biosimilarity studies.

Regulatory note: When used to support regulated workflows, qualification/validation of the digestion procedure and documentation of lot-specific characteristics (activity, formulation) are recommended. All such activities remain the responsibility of the user’s quality system. Item-specific pharmacopeial status is Not specified for this item; refer to CoA/Spec Sheet.

Physical Properties

Item-specific physical parameters (appearance, exact concentration, excipients, extinction coefficient, etc.): Not specified for this item; refer to CoA/Spec Sheet.

General/literature properties for Lys-C (context only; not item specs):

• State/form: Protein enzyme, typically supplied lyophilized or in aqueous buffer with stabilizers (varies by manufacturer).
• Solubility: Freely soluble in aqueous buffers; typical working buffers include 25–100 mM ammonium bicarbonate, Tris-HCl, or HEPES (pH ~7.5–9) (literature).
• pH activity profile: Broad activity in mildly basic conditions; often reported optimum around pH 8–9 (literature).
• Temperature profile: Active at 25–37°C; thermostability decreases at elevated temperatures; many workflows use 30–37°C (literature).
• Denaturant tolerance: Retains activity in high urea (commonly up to ~8 M), enabling proteolysis under denaturing conditions useful for proteomics (literature).
• Compatibility: Activity is reduced/inhibited by strong anionic detergents (e.g., SDS) unless mitigated by dilution or compatible surfactants; compatible with low concentrations of MS-compatible surfactants (literature).

Note: Exact values such as concentration, stabilizers, and formulation are item-specific and not provided here. Always confirm with the product’s CoA/SDS before experimental planning.

Quality and Grades

Item-specific quality statements:

• Grade/Purity: Recombinant; Suitable for mass spectrometry (MS); EnzymoPure.
• What this implies: MS-grade enzymes are prepared to minimize autolysis products and common protein contaminants that interfere with LC–MS workflows. EnzymoPure-format offerings generally indicate high enzymatic purity/specificity and low background that could complicate peptide identification. Exact specifications (activity units, host-cell protein/DNA, stabilizers) are Not specified for this item; refer to CoA/Spec Sheet.

Context and best practices (general):

• Recombinant production: Provides lot-to-lot consistency versus native extracts and reduces variability in cleavage specificity.
• MS suitability: Typically indicates low salt, low surfactant, and minimal non-volatile additives to ensure clean spectra and stable chromatographic baselines. Verify formulation with the CoA before direct injection workflows.
• Purity vs. performance: High-purity Lys-C reduces nonspecific proteolysis and background peptides; however, digestion performance also depends on buffer composition, pH, denaturants (e.g., urea), enzyme:substrate ratio, and incubation time.
• QC expectations (literature norms): Protease activity on model substrates, verified cleavage specificity (Lys-C rule), and low keratin/tryptic contaminants. Actual test methods and acceptance criteria vary by manufacturer and are not provided here.

Recommendation: For regulated or highly sensitive proteomics, retain the lot-specific CoA/SDS and validate digestion outcomes on in-house protein standards.

Reaction and Applications

Primary application: High-specificity proteolysis of proteins at the C-terminus of lysine (K) residues to generate peptide mixtures optimized for LC–MS/MS identification and quantification.

Key research uses (literature/general):

• Bottom-up proteomics: Lys-C digestion produces longer, less basic peptides than trypsin-only workflows; a common strategy is sequential Lys-C followed by trypsin to improve sequence coverage and reduce missed cleavages.
• Denaturing digests: Retained activity in urea (up to ~8 M) enables efficient proteolysis of difficult, aggregation-prone, or membrane-rich samples under denaturing conditions.
• Middle-down/top-down facilitation: Controlled, limited digestion to produce larger subunits for mapping PTMs and disulfides.
• Biopharmaceutical analytics: Peptide mapping of mAbs and other biologics for identity confirmation, PTM profiling (deamidation, glycation, oxidation), and lot comparability (process/QA contexts; not clinical use).

Practical considerations:

• Protein preparation: Reduce and alkylate to improve accessibility and peptide reproducibility. Remove or dilute inhibitors (e.g., SDS) and non-volatile salts prior to MS.
• Enzyme:substrate ratios (typical literature ranges): 1:50 to 1:200 (w/w), adjusted for protein complexity and denaturant concentration.
• Incubation: 30–37°C for 2–18 h depending on desired extent; monitor by SDS-PAGE or LC–MS sampling.
• Specificity notes: Cleavage efficiency can drop at Lys–Pro sites; high urea can mitigate structure-related hindrance (literature).

Reaction Conditions

The following are literature-based, general conditions for Lys-C digestion; they are provided as guidance and are not item-specific specifications.

Typical conditions (literature):

• Buffer: 25–100 mM ammonium bicarbonate, Tris-HCl, or HEPES, pH 8.0–8.5.
• Denaturants: 4–8 M urea supports robust activity for denaturing workflows.
• Temperature: 30–37°C.
• Enzyme:substrate (w/w): 1:50 to 1:200 for standard digests; adjust based on protein load, denaturant level, and desired speed.
• Incubation time: 2–18 h; monitor by SDS-PAGE or test LC–MS injections to determine endpoint.
• Reduction/alkylation: Reduce with DTT (5–10 mM, 30–60 min, 37°C) or TCEP (2–5 mM) and alkylate with IAA/CAA (10–25 mM, 20–30 min, RT in dark). Quench excess alkylating agent before digestion.
• Surfactants: Prefer MS-cleavable surfactants at ≤0.1% if needed; avoid high SDS or remove before digestion/MS (e.g., precipitation, SP3, SDC removal protocols).

Expected outcomes (literature):

• Cleavage C-terminal to Lys; fewer missed cleavages in high-urea denaturing workflows compared to trypsin alone.
• Peptide lengths skew slightly longer than tryptic peptides, beneficial for certain PTM localizations.

Note: Optimize conditions empirically for each sample matrix. Item-specific activity units, stabilizers, and exact formulations are Not specified for this item; consult the CoA/SDS.

Safety and Handling

Item-specific GHS classification, signal word, and H-statements: Not specified for this item; refer to SDS.

General enzyme handling guidance (literature/practice; defer to SDS for authoritative instructions):

• Potential hazards: Proteases can cause respiratory and skin sensitization upon repeated exposure. Avoid inhalation of aerosols/powders and contact with skin/eyes.
• PPE: Wear lab coat, disposable nitrile gloves, and chemical splash goggles. Use a certified biosafety cabinet or fume hood when manipulating dry powders or creating aerosols.
• Handling: Minimize aerosol generation. Keep containers tightly closed. Use only in well-ventilated areas. Avoid repeated freeze–thaw to preserve activity and limit formation of particulates.
• Incompatibilities: Strong acids/bases, oxidizing agents, organic solvents that denature proteins, and high concentrations of anionic detergents (e.g., SDS) can inactivate the enzyme (general guidance).
• First aid (overview): If inhaled, move to fresh air; seek medical attention if symptoms persist. In case of skin contact, wash with soap and water. For eye contact, rinse cautiously with water for several minutes and remove contact lenses if present/ easy to do; seek medical advice. If ingested, rinse mouth; do not induce vomiting; seek medical attention.
• Spill response: Avoid dust; gently cover with damp disposable towels, collect in a sealable container for disposal according to institutional and local regulations.
• Waste: Dispose in accordance with institutional biosafety and local environmental regulations.

Always consult the product SDS for definitive hazard classification and emergency measures.

Solvent Selection

This product is a protein enzyme used in aqueous buffers rather than an organic solvent. Solvent selection thus centers on buffer compatibility for proteolysis, cleanliness for MS, and compatibility with denaturants.

General/literature guidance (not item-specific):

• Primary media: Water-based buffers such as 25–100 mM ammonium bicarbonate (NH4HCO3), Tris-HCl, or HEPES at pH ~7.5–9. Ammonium bicarbonate is popular for LC–MS due to volatility.
• Denaturants/chaotropes: Lys-C remains active in high urea (commonly up to ~8 M), enabling digestion under denaturing conditions that unfold proteomes for improved accessibility.
• Surfactants: Use MS-compatible surfactants (e.g., acid-cleavable types) at low percentages if needed; strong SDS can inhibit activity and suppress MS unless removed prior to analysis.
• Reducing/alkylating agents: Reduction (e.g., DTT/TCEP) and alkylation (e.g., IAA/CAA) are typically performed prior to or during Lys-C digestion; these reagents are compatible with many proteomic buffers but must be quenched/removed as appropriate before LC–MS.
• Comparison with alternatives:
  • Ammonium bicarbonate: Volatile and MS-friendly; limited buffering above pH 8.3.
  • Tris-HCl: Strong buffering near pH 8, but non-volatile and can complicate MS; desalting recommended.
  • HEPES: Good pH control, but non-volatile; remove prior to MS.

Select buffers based on downstream MS needs, protein solubility, and desired pH; validate compatibility with your LC–MS method.

Storage and Reconstitution

Item-specific storage/shipping (from Product Data):

• Storage conditions: Store at −20°C. Avoid repeated freezing and thawing.
• Shipped in: Ice chest + ice pads.

General best practices for proteases (guidance; not item-specific specifications):

• Upon receipt: If lyophilized, allow container to equilibrate to room temperature in a desiccated environment before opening to minimize condensation. If in solution, keep on ice during handling.
• Reconstitution (if applicable and not otherwise specified by CoA): Reconstitute with chilled, LC–MS-grade water or a mildly acidic solution (e.g., 10–50 mM acetic acid or 1 mM HCl) to a convenient stock concentration. Gently mix—do not vortex vigorously.
• Aliquoting: Prepare single-use aliquots to avoid freeze–thaw cycles. Store aliquots at −20°C (short term) or −80°C (longer term), protected from repeated thermal cycling.
• Working solutions: Keep on ice during setup. Discard after the session or per established stability studies.
• Compatibility: Avoid buffers containing strong oxidants or high SDS that can denature/inactivate the enzyme. Verify buffer composition against your workflow and the product’s CoA.
• Stability: Actual shelf life, activity retention after reconstitution, and presence of stabilizers are Not specified for this item; refer to CoA/Spec Sheet.

Always consult the lot-specific CoA and SDS for definitive guidance on formulation, storage stability, and safe handling.

Structure and Identity

Brief description: Lys-C is a recombinant lysyl endoprotease, supplied in MS-grade (EnzymoPure) format for high-specificity proteolysis at the C-terminus of lysine residues in proteins prior to mass spectrometry.

• SKU: L1523246
• Product name: Lys-C (MS)
• CAS: 72561-05-8
• Grade/Purity (item-specific): Recombinant; Suitable for mass spectrometry (MS); EnzymoPure
• Category: Protein reagent (protease)
• Research use: For research use only

Structure identifiers for small molecules (SMILES, InChI, InChIKey) are not applicable to proteins and are not provided for this item.

Item-specific fields not provided:

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

Literature/general identity (for context; not item-specific specifications):

• Enzyme class: Serine endopeptidase; EC 3.4.21.50 (commonly known as Lysyl endopeptidase, “Lys-C”).
• Catalytic specificity: Cleaves on the C-terminal side of Lys residues in peptides/proteins; cleavage is significantly reduced when Lys is followed by Pro (literature).
• Typical polypeptide size: ~30 kDa single-chain enzyme (literature, source-dependent; recombinant constructs may vary).
• Common source organism (literature): Originally isolated from Lysobacter enzymogenes (historically Achromobacter).

Synthetic Utility

Although not a reagent for classical small-molecule synthesis, Lys-C serves as a biocatalyst for site-selective peptide bond hydrolysis and is highly valuable in analytical proteomics and protein chemistry.

General/literature utility:

• Site-selective cleavage: Generates peptides terminating at Lys (C-terminus), creating defined fragments for sequence analysis and MS/MS identification.
• Strategy design: Sequential digestion (Lys-C → trypsin) produces peptide sets that reduce missed cleavages and improve coverage for Lys-poor regions.
• Structural analysis: Limited proteolysis under native or mildly denaturing conditions can probe domain boundaries and folding states.
• Mapping PTMs: Produces peptide contexts that may be more suitable for localizing PTMs (e.g., phosphorylation near Arg-rich regions where trypsin alone may over-fragment).
• Protein engineering workflows: Controlled fragmentation for epitope mapping or to prepare subunits for advanced MS (native MS, top/middle-down; literature).

Constraints and considerations:

• Specificity caveats: Lys–Pro sites are resistant; conditions (denaturants, time) can be tuned to mitigate missed cleavages.
• Compatibility: Avoid strong anionic detergents and non-volatile salts prior to LC–MS; denaturant tolerance to urea provides flexibility versus trypsin-only methods.

Item-specific catalytic constants or activity units are Not specified for this item; refer to CoA/Spec Sheet.

Target Specificity

This section is typically used for antibodies or affinity reagents to list antigen/epitope specificity. Lys-C is a protease, not an affinity reagent; antigen/epitope specificity does not apply.

General enzymatic specificity (literature; not item-specific):

• Primary rule: Cleaves peptide bonds on the C-terminal side of lysine (K) residues.
• Notable exception: Cleavage efficiency is markedly reduced at Lys–Pro motifs.
• Substrate scope: Broad—acts on unfolded or denatured proteins; high tolerance to urea facilitates access to buried Lys sites.

Item-specific target or clone information: Not applicable.