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LysargiNase is a novel product of years of investigation.

Specially designed for proteolytic digestion of protein samples requiring identification by mass spectrometry.


 Characteristics


Mirror trypsin

Unlike trypsin, LysargiNase-generated peptides have N-terminal lysine or arginine residues and can be fragmented with b ion-dominated spectra.

lyserginase mirrors trypsin

Frequency distribution plots of cleavage sites identified by ion-trap CID from human proteome samples (n=4) digested with LysargiNase (n=1,917 sites) or trypsin (n=3,235 sites). Sequences were aligned at cleavage sites between P1 and P1′.

Complementary to trypsin

Complementary use of LysargiNase and trypsin increases proteome coverage.

 

Complementary use of LysargiNase and trypsin

 

Overlap of peptide sequences identified after LysargiNase or trypsin cleavage of proteomes.

Methylation insensitive

LysargiNase facilitates identification of peptides with mono-, di- and trimethylated arginine and lysine residues.

lyserginase-methylation-insensitiveStructural model depicting a possible interaction between a dimethylated lysine side chain and the LysargiNase S1′ pocket. LysargiNase accepts unmodified, mono- and dimethylated lysines at P1′ as shown in the structural model.

 Improved C-termini identification

LysargiNase generates significantly more protein C-terminal peptides compared with that by trypsin.

Improved C termini identification

Protein C- and N-terminal peptides as a proportion of all peptides identified by shotgun proteomics after trypsin (n = 4) or LysargiNase (n = 4) cleavage of proteome samples. Box plots: center lines, medians; box limits, 25th and 75th percentiles; whiskers extend to the 5th and 95th percentiles. Significance of differences was tested using the two-tailed Student′s t-test (***P < 0.001).

Improved phosphosite  identification

Phosphorylation motifs in trypsin- and LysargiNase-digested proteomes identified by the tool motif-x. Shown are the number and percentage of matching high-confidence phosphopeptides identified by MaxQuant at an FDR <0.01 with a localization probability >0.75. Phosphosites matching selected kinase specificity as extracted from the human protein reference database is shown as heat maps. Significance of differences was tested using Pearson’s χ2 test (*P < 0.05, **P < 0.01).

 

improved_phosphosite_identification

Active in various conditions

LysargiNase is active in a variety of conditions and solvents commonly  used  in  sample  preparation (5mM TCEP, 5% methanol, 5% acetonitrile, 0.8M urea, 0.1% RapiGest, 1% deoxycholate, 0.2% SDS, 1% NP-40 etc) and is suitable for in gel digestions.

lysarginase Active in various conditions

It is maximally active at temperatures up to 55°C, in the range of pH 6-9, and is reversible inhibited by 1, 10-phenanthroline, 5mM EDTA and other Ca++ and Zn++ chelating agents.

lysarginase Active in various conditions

Product information

Product description

LysargiNase kit is a novel product of many years of investigation that has been specially designed for proteolytic digestion of protein samples requiring identification by tandem mass spectrometry. It is provided lyophilized in two formats of 10×10μg or 10×100μg for use in 100 or 1000 reactions, respectively.

LysargiNase is a metalloproteinase found in the thermophilic archaea Methanosarcina acetivorans. Its stringent specificity cleaving before lysine and arginine makes it an attractive alternative for proteomics from the commonly used trypsin. The distribution of lysine and arginine residues in proteins is such that LysargiNase digestion yields peptides of molecular weights that can be analyzed by mass spectrometry. Unlike trypsin, LysargiNase-generated peptides have N-terminal lysine or arginine residues and can be fragmented with b ion–dominated spectra. This improves protein C-terminal–peptide identification and several arginine-rich phosphosite assignments. Notably, cleavage also occurs at methylated or dimethylated and trimethylated lysine and arginine, facilitating detection of these epigenetic modifications. Complementary use of LysargiNase and trypsin increases proteome coverage than just one of them individually [1].

LysargiNase has been manufactured to provide maximum specificity and proteolyitc activity. It is heterologously expressed in Escherichia coli following a highly optimized protocol that renders more stable and pure protein samples. Purification involves multiple chromatography steps including affinity and ion exchange chromatography giving protein samples more than 99% pure as estimated by SDS-PAGE.

LysargiNase is active in a variety of conditions and solvents commonly used in sample preparation (5mM TCEP, 5% methanol, 5% acetonitrile, 0.8M urea, 0.1% RapiGest, 1% deoxycholate, 0.2% SDS, 1% NP-40 etc) and is suitable for in gel digestions. It is maximally active at temperatures up to 55°C, in the range of pH 6-9, and is reversibly inhibited by 1, 10- phenanthroline, ethylenediaminetetraacetic acid (EDTA) and other Ca++ and Zn++ chelating agents [1, 2].

Components

• LysargiNase, Enzyme

• LysargiNase Reaction Buffer

Preparation Instructions

Ultrapure water (18MΩ⋅cm or equivalent) is recommended for preparation of all reagents.

LysargiNase, Enzyme – each vial contains 10-100μg of enzyme as indicated. Rapidly spin down each vial and then reconstitute the lyophilized protein with 10μl or 100μl of ultrapure water, respectively. Once in solution (50mM HEPES, 5mM CaCl2, pH 7.5) store at – 20°C and avoid repeated freeze thaw steps.

LysargiNase, Reaction Buffer – Solution of 50mM HEPES, 5mM CaCl2, pH 7.5.

Storage/Stability – It is recommended to store the kit at -20ºC. The protease is stable to use for up to 1 month after reconstituting the enzyme in water.

Procedure

For peptide or protein digestion in solution or in gel, a ratio of between 1:100 and 1:20 (w/w) of enzyme to substrate is recommended.

References

1. Tallant C, García-Castellanos R, Seco J, Baumann U, Gomis-Rüth FX (2006) Molecular analysis of ulilysin, the structural prototype of a new family of metzincin metalloproteases. J Biol Chem. 281(26):17920-17928

2. Huesgen PF, Lange PF, Rogers LD, Solis N, Eckhard U, Kleifeld O, Goulas T, Gomis-Rüth FX, Overall CM (2015) LysargiNase mirrors trypsin for protein Cterminal and methylation-site identification. Nat Methods 12(1):55-58

Price and Availability

Dear Customer,

thanks for your interest. We have actually reached an agreement with EMD/Millipore to take over marketing and distribution of lysargiNase. Please, get in touch with Amy Villanueva (amy.villanueva@emdmillipore.com) for prices, orders and delivery schedules.

Best regards,

The LysargiNase Team

P.S. Please, send an e-mail to lysarginase@ibmb.csic.es if you encounter any difficulties.

References

Tallant, C. et al. J. Biol. Chem. 281, 17920–17928 (2006).

Garcia-Castellanos. R., et al. Arch. Biochem. Biophys. 457, 57–72 (2007).

Tallant, C. et al. Biol. Chem. 388, 1243–1253 (2007).

Gomis-Rüth, F.X. Crit. Rev. Biochem. Mo. Biol. 43, 319–345 (2008).

Tallant, C. et al. J. Biol. Chem. 285, 3951–13957 (2010).

Huesgen, P.F. et alNat. Methods 12, 55–58 (2015).

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