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Lysyl endopeptidase

Lysyl endopeptidase is a protease enzyme that cleaves peptide bonds on the carboxyl side of lysine residues.
It is commonly used in protein sequencing and analysis, as well as in research involving protein structure and function.
PubCompare.ai's AI-driven platform can help optimize your Lysyl endopeptidase research by locating the best protocols from literature, pre-prints, and patens, and enhancing your accuracy with intelligent protocol comparisons.
Leverge this cutting-edge technology to streamline your research process and achieve superior results.

Most cited protocols related to «Lysyl endopeptidase»

1
SILAC and Label-free Mass Spectrometry Workflow
Cited 31 times
After processing raw files with the in house–developed software MaxQuant (version 1.0.12.36 or 1.0.13.12; Cox and Mann, 2008 (link)), data were searched against the human database concatenated with reversed copies of all sequences (Peng et al., 2003 (link)) and supplemented with frequently observed contaminants (porcine trypsin, achromobacter lyticus lysyl endopeptidase, and human keratins) using MASCOT (version 2.2.0; Matrix Science). For the analysis of pericentrin experiments, the mouse pericentrin sequence was added to the database. Carbamidomethylated cysteins were set as fixed, oxidation of methionine, and N-terminal acetylation as variable modification. Mass deviation of 0.5 D was set as maximum allowed for MS/MS peaks, and a maximum of two missed cleavages were allowed. Maximum false discovery rates (FDRs) were set to 0.01 both on peptide and protein levels. Minimum required peptide length was six amino acids.
Quantification of proteins in SILAC experiments was performed using MaxQuant (Cox and Mann, 2008 (link)). Methionine oxidations and acetylation of protein N termini were specified as variable modifications and carbamidomethylation as fixed modification. Maximum peptide charge was set to 6. SILAC settings were adjusted to doublets, and Lys0 and Lys8 were selected as light and heavy label, respectively. Peptide and protein FDRs were set to 0.01. The maximum PEP was set to 1, and six amino acids were required as minimum peptide length. Only proteins with at least two peptides (thereof one uniquely assignable to the respective protein group) were considered as reliably identified. Unique and razor peptides were considered for quantification with a minimum ratio count of 2. Forward and reverse experiments were analyzed together and specified as QUBICH and QUBICL in the experimentalDesign.txt. Ratios of the reverse experiment QUBICL were inverted. Specific interaction partners in SILAC experiments were determined by a combination of ratio and ratio significance calculated by MaxQuant. The p-value for the significance of enrichment had to be <0.01 in both the forward and reverse experiment. The provided R script QUBIC-SILAC.R was used to plot all identified proteins according to their ratios in the forward and reverse experiment and mark specific interaction partners (http://www.r-project.org).
Label-free quantification was performed with MaxQuant (see Supplemental data). Methionine oxidations and acetylation of protein N termini were specified as variable modifications and carbamidomethylation as fixed modification. Maximum peptide charge was set to 6. SILAC settings were set to singlets. Peptide and protein FDRs were set to 0.01. The maximum PEP was set to 1, and six amino acids were required as minimum peptide length. Only proteins with at least two peptides (thereof one uniquely assignable to the respective protein group) were considered as reliably identified. Label-free protein quantification was switched on, and unique and razor peptides were considered for quantification with a minimum ratio count of 1. Retention times were recalibrated based on the built-in nonlinear time-rescaling algorithm. MS/MS identifications were transferred between LC-MS/MS runs with the “Match between runs” option in which the maximal retention time window was set to 2 min. The quantification is based on the extracted ion current and is taking the whole three-dimensional isotope pattern into account. At least two quantitation events were required for a quantifiable protein. Every single experiment/raw file was annotated as a separate experiment in experimentalDesign.txt. Control experiments were named Control1, Control2, and Control3. Pull-downs were named with the specific bait name and the replicate number. Identification of specific interaction partners was determined using the MaxQuant-based program QUBICvalidator. The proteinGroups.txt file was loaded (Load – Generic), and a group file template, Groups.txt, was generated (Processing – Groups – Write group file template). Replicates were grouped using one unique name in Groups.txt. The file was then loaded into QUBICvalidator (Processing – Groups – Load groups). Subsequently, results were cleaned for reverse hits and contaminants (Processing – Filter – Filter category – Reverse = + and Contaminant = +). Positive intensity values were logarithmized (Processing – Transformation – LOG – Log2). Signals that were originally zero were imputed with random numbers from a normal distribution, whose mean and standard deviation were chosen to best simulate low abundance values below the noise level (Processing – Imputation – Replace missing values by normal distribution – Width = 0.3; Shift = 1.8). Significant interactors were determined by a volcano plot-based strategy, combining t test p-values with ratio information. The standard equal group variance t test was applied (Processing – Testing – Two groups). Significance lines in the volcano plot corresponding to a given FDR were determined by a permutation-based method (Tusher et al., 2001 (link)). The pull-down was selected as Group1 and the control as Group2. Threshold values (= FDR) were selected between 0.1 and 0.001 and SO values (= curve bend) between 0.5 and 2.0. The resulting table was then exported (Export – Tab separated). The second tab (Table S1 and Table S2) was selected, and values saved with the same file name were supplemented with “_sup” (e.g., Exp.txt → Exp_sup.txt). Results were then plotted using the open source statistical software R and the provided script QUBIC-LABELFREE.R. In the beginning of the script, Exp.txt and Exp_sup.txt have to be replaced with the real file names. Dynamic experiments were plotted using the script QUBIC-LABELFREE_dynamic.R. Significant TREX and TACC3 interactors were clustered using Genesis (Sturn et al., 2002 (link)).
A detailed step by step protocol and the raw data and programs associated with this manuscript may be downloaded from https://proteomecommons.org/tranche, launching Tranche, choosing “Open By Hash”, and entering the following hash: iNYsECWFuN0KDV0Q8QoE3uXxRGuBiCo5+iwydOM7h29jlyPv+Xv4+1piRkFr+mcnsy+eErYIvmcRQf9ZU/l5lxQYNQYAAAAAAABFCA==
Hubner N.C., Bird A.W., Cox J., Splettstoesser B., Bandilla P., Poser I., Hyman A, & Mann M. (2010). Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. The Journal of Cell Biology, 189(4), 739-754.
Publication 2010
2
Tissue Homogenization and Protein Extraction
Cited 20 times

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Publication 2016
bicinchoninic acid Brain Buffers Cold Temperature Dithiothreitol Freezing Iodoacetamide lysyl endopeptidase Nucleic Acids Peptide Hydrolases Peptides Phosphoric Monoester Hydrolases Promega Proteins SDS-PAGE Sep-Pak C18 Stainless Steel Tissues Trypsin Urea Vacuum
3
SILAC-Based HeLa Cell Proteome Analysis
Cited 19 times
Hela cells were grown over 8 passages in High Glucose DMEM [12.43 g/l Dulbecco’s Modified Eagle’s Medium (Caisson Laboratories Inc., North Logan, UT, USA), 4.5 g/l D-(+)-Glucose anhydrous (Fluka, Buchs, Switzerland), 30 mg/l Glycine (Fluka)] supplemented with either light (Sigma-Aldrich) or heavy (13C 15N, Sigma-Aldrich) isotope-labeled lysine and arginine at 37°C and 5% CO2.
Cells were harvested at 80% confluency by trypsinization, washed three times with ice cold PBS (GIBCO (Invitrogen), Paisley, UK) and the cell number was determined using a Neubauer chamber. Hela cells were spun down at 300 ×g and resuspended in one cell pellet volume PBS. Two pellet volumes of 8 M Urea (Sigma-Aldrich, Buchs, Switzerland) containing 50 mM ammonium bicarbonate (Sigma-Aldrich) and 0.1% RapiGest (Waters, Baden, Switzerland) were thoroughly mixed with the resuspended cells. Subsequent to sonication (80% amplitude, 0.6 cycle, 1 min) cell debris was spun down at 16000×g.
The protein concentration of the lysate was measured by BCA assay (bicinchoninic acid, Thermo Scientific, Reinach, Switzerland). Proteins were reduced with 5 mM TCEP (tris(2-carboxyethyl)phosphine, Thermo Scientific) at 37°C for 15 min and alkylated with 10 mM iodoacetamide (Sigma-Aldrich) for 30 min in the dark. Proteins were first digested with lysyl endopeptidase (Wako Chemicals, Neuss, Germany) at an enzyme – substrate ratio of 1 to 50 (w/w) at 35°C for 2 hours. After dilution with 50 mM ammonium bicarbonate to 0.8M urea trypsin (Promega) was added at the same ratio. Tryptic digestion was carried out overnight at 37 °C. Peptides were acidified with 1% trifluoroacetic acid (TFA, Thermo Scientific) and purified by solid-phase extraction using C18 cartridges (Sep-Pak, Waters). The SPE eluate was evaporated to dryness and reconstituted in 3% acetonitrile (Thermo Scientific) and 0.2% formic acid (Sigma-Aldrich).
Escher C., Reiter L., MacLean B., Ossola R., Herzog F., Chilton J., MacCoss M.J, & Rinner O. (2012). Using iRT, a normalized retention time for more targeted measurement of peptides. Proteomics, 12(8), 1111-1121.
Publication 2012
acetonitrile ammonium bicarbonate Arginine bicinchoninic acid Biological Assay Cells Cold Temperature Digestion Eagle Enzymes formic acid Glucose Glycine HeLa Cells Iodoacetamide Isotopes Lysine lysyl endopeptidase Peptides phosphine Promega Proteins Solid Phase Extraction Technique, Dilution TNFSF14 protein, human Trifluoroacetic Acid tris(2-carboxyethyl)phosphine Tromethamine Trypsin Urea
4
Profiling Plasma Proteome Variations
Cited 12 times
Plasma samples were heat-treated and then reacted with lysyl-endopeptidase. Plasma proteins were subsequently digested by trypsin and desalinated. These plasma samples were analyzed in triplicate by LC-tandem MS. Three mass spectrometers were used to process 501 plasma samples from 501 individuals; 233 samples by Thermo Scientific Orbitrap Fusion, 156 samples by Thermo Scientific Elite, and 112 samples by Thermo Scientific Q Exactive. Peptide identification from mass spectra was obtained using the SequestHT and Mascot search engines with the UniProt human proteome data set from April 2014 as reference protein sequences. These peptide identification results were integrated using Proteome Discoverer1.4. The abundance of a specific protein in the 501 samples was calculated as the fraction of samples in which the protein was successfully identified. To identify peptides resulting from non-synonymous genomic variations, we created a data set of protein sequences containing alternative alleles found in at least 5% of the ToMMo 1KJPN cohort. This database was also searched for peptide sequences harboring alternative alleles (amino acids). All reference and alternative peptide sequences observed in our plasma proteome analysis are listed on the peptide table page.
Tadaka S., Saigusa D., Motoike I.N., Inoue J., Aoki Y., Shirota M., Koshiba S., Yamamoto M, & Kinoshita K. (2017). jMorp: Japanese Multi Omics Reference Panel. Nucleic Acids Research, 46(Database issue), D551-D557.
Publication 2017
Alleles Amino Acids Amino Acid Sequence ANP32B protein, human Genome Homo sapiens lysyl endopeptidase Mass Spectrometry Peptides Plasma Plasma Proteins Proteins Proteome Staphylococcal Protein A Trypsin
5
Quantitative Proteomic Analysis of iPSCs
Cited 9 times
For protein extraction, iPSC cell pellets were washed with ice cold PBS and redissolved immediately in 200 μl of lysis buffer (8 m urea in 100 mm triethyl ammonium bicarbonate (TEAB)) and mixed at room temperature for 15 min. Cellular DNA was sheared using ultrasonication (6 × 20 s on ice). The proteins were reduced using tris-carboxyethylphosphine TCEP (25 mm) for 30 min at room temperature, then alkylated in the dark for 30 min using iodoacetamide (50 mm). Total protein was quantified using the EZQ assay (Thermo Fisher Scientific, Waltham, MA). For the first digestion with mass spectrometry grade lysyl endopeptidase, Lys-C (Wako, Japan), the lysates were diluted 4-fold with 100 mm TEAB then further diluted 2.5-fold before a second digestion with trypsin. Lys-C and trypsin were used at an enzyme to substrate ratio of 1:50 (w/w). The digestions were carried out overnight at 37 °C, then stopped by acidification with trifluoroacetic acid (TFA) to a final concentration of 1% (v:v). Peptides were desalted using C18 Sep-Pak cartridges (Waters, Millford, MA) following manufacturer's instructions.
For tandem mass tag (TMT)-based quantification, the dried peptides were re-dissolved in 100 mm TEAB (50 μl) and their concentration was measured using a fluorescent assay (CBQCA, Thermo Fisher Scientific). For each 10-plex TMT batch 100 μg of peptides from each cell line to be compared, in 100 μl of TEAB, were labeled with a different TMT tag (20 μg/ml in 40 μl acetonitrile) (Thermo Fisher Scientific), for 2 h at room temperature. After incubation, the labeling reaction was quenched using 8 μl of 5% hydroxylamine (Thermo Fisher Scientific) for 30 min and the different cell lines/tags were mixed and dried in vacuo.
The TMT samples were fractionated using off-line, high-pH reverse-phase (RP) chromatography: samples were loaded onto a 4.6 × 250 mm Xbridge BEH130 C18 column with 3.5-μm particles (Waters). Using a Dionex bioRS system, the samples were separated using a 25-min multistep gradient of solvents A (10 mm formate at pH 9) and B (10 mm ammonium formate pH 9 in 80% acetonitrile), at a flow rate of 1 ml/min. Peptides were separated into 48 fractions, which were consolidated into 24 fractions. The fractions were subsequently dried and the peptides re-dissolved in 5% formic acid and analyzed by LC-MS/MS.
Brenes A., Hukelmann J., Bensaddek D, & Lamond A.I. (2019). Multibatch TMT Reveals False Positives, Batch Effects and Missing Values. Molecular & Cellular Proteomics : MCP, 18(10), 1967-1980.
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Publication 2019
3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde acetonitrile Biological Assay Buffers Cell Lines Cells Chromatography, Reverse-Phase Cold Temperature Digestion Enzymes formate formic acid formic acid, ammonium salt Hydroxylamine Induced Pluripotent Stem Cells Iodoacetamide lysyl endopeptidase Mass Spectrometry Pellets, Drug Pepsin A Peptides Proteins Solvents Tandem Mass Spectrometry triethylammonium bicarbonate Trifluoroacetic Acid tris(2-carboxyethyl)phosphine Tromethamine Trypsin Urea

Most recents protocols related to «Lysyl endopeptidase»

1
On-bead Protein Digestion for LC-MS/MS
On-bead digestion in preparation for LC-MS/MS was performed following an established protocol.[49 (link)] Digestion buffer (50 mM NH4HCO3) was added to the beads, and the mixture was treated with 1 mM dithiothreitol (DTT) at room temperature for 30 minutes, followed by 5 mM iodoacetimide (IAA) at room temperature for 30 minutes in the dark. Proteins were then digested overnight with 2 μg of lysyl endopeptidase (Wako) at room temperature and further digested overnight with 2 μg trypsin (Promega) at room temperature. Resulting peptides were desalted with HLB column (Waters) and were dried under vacuum.
DiCandia M.A., Edwards A.N., Alcaraz Y.B., Monteiro M.P., Lee C.D., Vargas Cuebas G., Bagchi P, & McBride S.M. (2024). A conserved switch controls virulence, sporulation, and motility in C. difficile. PLOS Pathogens, 20(5), e1012224.
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Publication 2024
2
Bead-based Protein Digestion Protocol
Digestion was performed on beads using a digestion buffer containing 50 mM NH4HCO3. The beads were then treated with 1 mM DTT at 25 °C for 30 min, followed by addition of 5 mM iodoacetamide (IAA) at 25 °C for 30 min in the dark. Lysyl endopeptidase (Wako) was added to the mixture at a 1:50 (w/w) enzyme to protein ratio and digestion proceeded at 25 °C overnight. Samples were further digested overnight with 1:50 (w/w) trypsin (Promega) at 25 °C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.
de Amorim J.L., Leung S.W., Haji-Seyed-Javadi R., Hou Y., Yu D.S., Ghalei H., Khoshnevis S., Yao B, & Corbett A.H. (2024). The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops). The Journal of Biological Chemistry, 300(2), 105646.
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Publication 2024
3
Comprehensive Serum Proteome and Glycoproteome Analysis
Protein extraction and digestion was performed as previously described [30 (link)]. In brief, the serum protein was denatured in 8 M urea/ 50 mM NH4HCO3, then reduced with DTT, alkylated with IAM, digested with lysyl endopeptidase (Promega; Madison, WI, USA) for 4 h at 37 °C, and then incubated with trypsin (Promega) overnight at 37 °C with shaking. The digested peptides were acidified with 10% trifluoroacetic acid to pH < 3, collected by centrifugation and purified using Oasis HLB cartridges (Waters; Milford, MA, USA). Desalted peptides were subjected to LC–MS/MS analysis for proteomics. N-glycopeptides were enriched by MAX column (Waters) using desalted peptides, and subjected to LC–MS/MS analysis for glycoproteomics as described previously [35 (link)].
Li Z., Zhang N., Dong Z., Wang X., Zhou J., Gao J., Yang Y., Li J., Guan F., Zhou Y, & Tan Z. (2024). Integrating transcriptomics, glycomics and glycoproteomics to characterize hepatitis B virus-associated hepatocellular carcinoma. Cell Communication and Signaling : CCS, 22, 200.
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Publication 2024
4
Vacuolar Protein Digestion and Purification
Cross-linked vacuoles were digested in solution. Proteins were denatured by incubation in 8 M urea in 50 mM tetraethylammonium bromide (TEAB), reduced with 5 mM dithiothreitol (DTT) for 60 min at 37 °C and alkylated with 40 mM chloroacetamide at room temperature for 30 min in the dark. Proteins were digested with Lysyl endopeptidase C (Wako) at an enzyme-to-protein ratio of 1:75 (w/w) at 37 °C for 4 h. After diluting with 50 mM TEAB to a final concentration of 2 M urea, the digestion was continued with trypsin (Serva) at an enzyme-to-protein ratio of 1:100 (w/w) at 37 °C overnight. Peptides were desalted with Sep-Pak C18 cartridges (Waters) and dried in a SpeedVac.
Klössel S., Zhu Y., Amado L., Bisinski D.D., Ruta J., Liu F, & González Montoro A. (2024). Yeast TLDc domain proteins regulate assembly state and subcellular localization of the V-ATPase. The EMBO Journal, 43(9), 1870-1897.
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Publication 2024
5
Protein Sample Preparation for MS

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Publication 2024

Top products related to «Lysyl endopeptidase»

1
Lysyl endopeptidase by Fujifilm
Sourced in Japan, Germany, United States
Lysyl endopeptidase is a proteolytic enzyme that specifically cleaves peptide bonds on the carboxyl side of lysine residues in proteins. It is commonly used in laboratory settings for protein analysis and modification.
2
Trypsin by Promega
Sourced in United States, Germany, United Kingdom, China, Japan, France, Switzerland, Sweden, Italy, Netherlands, Spain, Canada, Brazil, Australia, Macao
Trypsin is a serine protease enzyme that is commonly used in cell culture and molecular biology applications. It functions by cleaving peptide bonds at the carboxyl side of arginine and lysine residues, which facilitates the dissociation of adherent cells from cell culture surfaces and the digestion of proteins.
3
Lysyl endopeptidase lys c by Fujifilm
Sourced in Japan, United States, Germany
Lysyl endopeptidase (Lys-C) is a protease enzyme that cleaves peptide bonds on the carboxyl side of lysine residues. It is used in protein analysis and sample preparation for mass spectrometry applications.
4
Lys c by Fujifilm
Sourced in Japan, United States, Germany
Lys-C is a protease enzyme used in proteomics research. It selectively cleaves proteins at the carboxyl side of lysine residues, which can be useful for generating peptide fragments for analysis.
5
Sequencing grade modified trypsin by Promega
Sourced in United States, United Kingdom, Germany, France, Switzerland, Japan, China, Sweden
Sequencing grade modified trypsin is a protease enzyme used for the digestion of proteins prior to mass spectrometry analysis. It is designed to provide consistent, high-quality peptide digestion for protein identification and characterization.
6
Sep pak c18 column by Waters Corporation
Sourced in United States, United Kingdom, Australia
The Sep-Pak C18 column is a solid-phase extraction (SPE) cartridge used for sample preparation in analytical chemistry. It contains a silica-based stationary phase with C18 (octadecyl) functional groups, which are effective for the retention and separation of a wide range of organic compounds. The column is designed to facilitate the extraction, purification, and concentration of analytes from complex sample matrices.
7
Sequencing grade trypsin by Promega
Sourced in United States, Germany, United Kingdom, Italy, France, Spain, Switzerland
Sequencing grade trypsin is a proteolytic enzyme used to cleave peptide bonds in protein samples, primarily for use in protein sequencing applications. It is purified to ensure high-quality, consistent performance for analytical processes.
8
Iodoacetamide by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe, Italy, France, Macao, China, Canada, Australia, Switzerland, Spain
Iodoacetamide is a chemical compound commonly used in biochemistry and molecular biology laboratories. It is a reactive compound that selectively modifies cysteine residues in proteins, thereby allowing for the study of protein structure and function. Iodoacetamide is often used in sample preparation procedures for mass spectrometry and other analytical techniques.
9
Hlb column by Waters Corporation
Sourced in United States, Ireland
The HLB column is a type of solid-phase extraction (SPE) column used for sample preparation in analytical chemistry. The column contains a hydrophilic-lipophilic balanced (HLB) sorbent material that is effective in retaining and separating both polar and non-polar compounds from liquid samples. The core function of the HLB column is to facilitate the extraction, concentration, and purification of a wide range of analytes prior to instrumental analysis.
10
Q exactive mass spectrometer by Thermo Fisher Scientific
Sourced in United States, Germany, China, United Kingdom, Japan, Denmark, Uruguay
The Q Exactive mass spectrometer is a high-resolution, accurate-mass (HRAM) instrument designed for advanced proteomics, metabolomics, and small molecule applications. It combines a quadrupole mass filter with a high-field Orbitrap mass analyzer to provide precise mass measurements and high-quality data.

More about "Lysyl endopeptidase"

Lysyl endopeptidase, also known as Lys-C, is a powerful protease enzyme that plays a crucial role in protein sequencing, analysis, and research involving protein structure and function.
This enzyme specifically cleaves peptide bonds on the carboxyl side of lysine residues, making it a valuable tool for protein identification and characterization.
Trypsin, another commonly used protease, cleaves peptide bonds on the carboxyl side of arginine and lysine residues.
However, Lysyl endopeptidase offers several advantages over trypsin, as it is more selective and can provide more detailed information about protein sequences.
Sequencing grade modified trypsin and sequencing grade trypsin are also important tools in protein analysis, as they are optimized for high-quality protein sequencing.
Similarly, Sep-Pak C18 columns and HLB columns are used for sample preparation and purification, helping to ensure accurate and reliable results.
The use of iodoacetamide in protein analysis is crucial for the alkylation of cysteine residues, preventing the formation of disulfide bonds and ensuring complete denaturation of the protein.
The Q Exactive mass spectrometer is a powerful instrument that is often used in conjunction with these protease enzymes and sample preparation techniques, enabling researchers to achieve high-resolution, accurate mass measurements of peptides and proteins.
By leveraging the insights and tools provided by PubCompare.ai's AI-driven platform, researchers can streamline their Lysyl endopeptidase-based studies, locate the best protocols from literature, pre-prints, and patents, and enhance the accuracy of their results through intelligent protocol comparisons.
This cutting-edge technology can help researchers achieve superior outcomes in their protein research endeavors.