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Pierce Lys N Protease, MS Grade


fast-digesting alternative to trypsin that yields complementary peptides.

Pierce Lys-N Protease, MS Grade

Thermo Scientific Pierce LysN Protease, MS Grade, is highly purified native Lys-N protease that has been validated for maximum activity and stability in proteomic applications.

Thermo Scientific Pierce LysN is a mass spectrometry (MS)-grade zinc metalloprotease derived from Grifola frondosa. The native Pierce LysN Protease has been highly purified to improve stability, specific activity, and cleavage selectivity. Unlike trypsin, LysN protease cleaves at the amino-terminus of lysine residues. As a result, the peptides generated by LysN are longer than those generated by trypsin and have more prevalent charged amino terminal peptide fragments. Additionally, LysN protease is more promising for epigenetic MS applications than LysC or trypsin because it is capable of cleaving methylated lysines [1]. The thermostability and chemical compatibility with denaturants make LysN protease ideal for digestion of complex protein samples for “shotgun” proteomics.

Highlights:

  • Thermostable – provides better digestion at higher temperatures (e.g., 50°C) in less time
  • Complementary to tryptic digests cleaves the opposite side of lysine from trypsin
  • High purity – no additional (off-target) protease activity detected
  • N-terminal lysine cleavage specificity – at least 90% for a complex protein sample
  • Versatile – enzyme is effective at many temperatures and in denaturing conditions
  • Stable – provided in a lyophilized format

Applications:

  • Improved sequence coverage of protein digests
  • De novo sequencing with collision induced dissociation (CID)
  • Epigenetic studies
  • In-solution digestion of proteins

Product Details:

LysN protease is active over a wide range of temperatures and denaturing conditions. Efficient protein digestion can be completed in 2 hours at 50°C or 4 hours at 37°C, however, digestion is possible at both lower and higher temperatures. LysN also remains active under moderate denaturing conditions including 0.1% SDS, 6M urea or heating to 70°C. Maximal LysN activity occurs at pH 7 to 9. The lyophilized enzyme is stable for 2 years when stored at -20°C, and reconstituted stock solutions of LysN are stable at -80°C for 2 years or -20°C for one year without significant loss in activity.

Cleavage sites for Lys-N and trypsin proteases
Cleavage site differences between LysN and trypsin.

 

Complementary cleavage by Lys-N and trypsin proteases
Complementary cleavage by Lys-N and Trypsin proteases for mass spectrometry. Comparisons of in-solution digestion and sequence coverage by Pierce LysN Protease (Part No. 90300) and Pierce Trypsin Protease (Part No. 90057). A proportional Venn diagram of protein identifications from two A549 digestions using LysN or trypsin (A). Samples (50µg) were prepared with the Pierce Mass Spec Sample Prep Kit for Cultured Cells and digested with LysN at a 1:75 enzyme:protein ratio and incubated at 37oC for 4 hours, or digested with trypsin at a 1:50 enzyme:protein ration and incubated at 37oC overnight. Digested sample peptides (0.5µg) were analyzed by nano LC-MS on an Orbitrap XL mass spectrometer. CID MS/MS data were searched using Sequest HT search engine. 986 proteins were identified from both LysN and trypsin digests whereas 1190 proteins were identified from LysN and 1156 were identified from trypsin at 1% FDR. Sequence coverage differences for LysN vs. trypsin and their merged independent digestions of two different proteins are shown (B).

 

Exceptional digestion performance by Pierce Lys-N Protease
Exceptional digestion performance with Thermo Scientific Pierce LysN Protease, MS Grade. Base peak LC/MS chromatogram of an A549 cell lysate sample digested with Pierce LysN protease. The sample (50µg) was prepared with the Pierce Mass Spec Sample Prep Kit for Cultured Cells and digested in solution with LysN at a 1:75 protein:enzyme ratio and incubated at 37°C for 4 hours. Digested sample peptides (0.5µg) were separated using nanoflow high-pressure liquid chromatography and analyzed by a Thermo Scientific Orbitrap XL mass spectrometer.

 

Percent sequence coverage of selected proteins in an A549 cell lysate upon in-solution digestion with Lys-N or trypsin.  The total (Merged) sequence coverage represents the combined coverage of separate LysN and trypsin digestions followed by LC-MS/MS analysis with CID.
Protein Lys-N Trypsin Merged
Moesin 21% 24% 37%
Src substrate cortactin 25% 10% 30%
Glutamate dehydrogenase 1 16% 12% 28%
Epoxide hydrolase 1 13% 14% 23%

General References:

  1. Taouatas N., et al. (2010) Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. J Proteome Res. 9(8):4282-8.
  2. Gershon, P., (2014) Cleaved and missed sites for trypsin, Lys-C, and Lys-N can be predicted with high confidence on the basis of sequence context. J. Proteome Res. 13(2):702-9.
  3. Nonaka T, J., et al. (1998) Kinetic characterization of lysine-specific metalloendopeptidases from Grifola frondosa and Pleurotus ostreatus fruiting bodies. Biochemistry 124(1):157-62.

Related Products:

Pierce Trypsin Protease, MS Grade
Mass spectrometry reagents and kits
Detergent Removal Products
Mass Spec Sample Prep Kit for Cultured Cells


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
90300 Lys-N Protease, MS Grade
Formulation: Purified Lys-N, lyophilized

20μg Product Instructions for product #90300 Lys-N Protease, MS Grade MSDS for product #90300 Lys-N Protease, MS Grade Certificate of Analysis for product #90300 Lys-N Protease, MS Grade $250.00

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90301 Lys-N Protease, MS Grade
Formulation: Purified Lys-N, lyophilized

5 x 20μg Product Instructions for product #90301 Lys-N Protease, MS Grade MSDS for product #90301 Lys-N Protease, MS Grade Certificate of Analysis for product #90301 Lys-N Protease, MS Grade $900.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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