Life Science
Life Science
Lysyl Endopeptidase, Mass Spectrometry Grade
(Wako Catalog No. 125-05061 (5 x 20μg))
Among the most important techniques in proteome analyses is the in-gel digestion of protein spots/bands that have been resolved by electrophoresis using digestive enzymes, such as trypsin and lysyl endopoptidase. Proteins can be identified by mass spectrometry analysis of the peptides produced by in-gel digestion, and further information regarding post-translational modifications can be obtained.
Lysyl Endopeptidase, Mass Spectrometry Grade is a freeze dried product that retained sufficient activity for in-gel digestion and packed in very small quantities for convenience purposes.
product
[Features]
1. High specificity and efficiency of protein digestion allow for easy database searches by peptide mass.
2. Improved cleavage at lysine residue and increase in the number of peptides are obtained by combination with trypsin.
3. Packed in very small quantities according to the amounts used so that sufficient activity for in-gel digestion may be retained.
Comparison of In-gel Digestion Using Trypsin (Tp), Lysyl Endopeptidase (Lep) and Lep Combined with Tp (Lep +Tp)
BSA band (100ng) resolved by SDS-PAGE was in-gel digested with Tp, Lep and Lep +Tp and analyzed by MALDI-TOFMS. The figure shows the individual mass spectra. The evaluation of these peptidases is summarized in the table.
[Table: Comparison of Trypsin (Tp), Lysyl Endopeptidase (Lep) and Lep +Tp]
Tp
Lep
Lep + Tp
Cleavage site
C terminal of Arg and Lys
C terminal of Lys
C terminal of Arg and Lys
Missed cleavage (Rates of missed cleavage)*
Many (8%)
Very few (0%)
Few (3%)
No. of identified peptides
17
19
22
These results indicate there are very few missed cleavages obtained by Lep digestion. When Tp is used concomitantly with Lep, missed cleavages decrease and the number of identified peptides increase compared to when only Tp is used.
* The value resulted from subtracting the coverage obtained when database searches were performed with Missed cleavage 0 from that obtained when performed with Missed cleavage 1. "Coverage" is the percentage of peptides obtained after in-gel digestion in the whole sequence.
table
[References]
(1) Wada, Y., and Kadoya, M.: J. Mass Spectrom., 38, 117 (2003).;
(2) Shevchenko, A., Wilm, MM., Vorm, O., and Mann., M.: Anal. Chem., 68, 850 (1996).

Endoproteinase Asp-N, Sequence grade
(Wako Catalog No. 056-05921 (2μg))
  • Origin: Pseudomonas fragi, mutant
  • Preparation: Lyophilized
  • Specificity: Cleave on the N-terminal side of Aspartic acid and Cysteic acid; after alkylation of Cys: only cleavage on Asp.
  • Specific activity: Indicated on each label (at 37°C with azocoll as substrate).
  • Purity: min. 90% (SDS-PAGE)
  • Performance: Cleavage after 1hr: min. 90% (with glucagon as substrate).
product Glucagon:
   
H-S-Q-G-T-F-T-S-D(9)-Y-S-K-Y-L-D(15)-S-R-R-A-Q
   -D(21)-F-V-Q-W-L-M-N-T
    1. Asp (15)-Gln (20);
    2. His (1)-Ser (8);
    3. Asp (9)-Leu (14);
    4. Asp (21)-Thr (29)
Digest:
  100 μg Glucagon + 10 μg Endoproteinase Asp N Sequencing grade in 100 μL Sodium Phosphate Buffer (50 mmol/L, pH 8.0) 1h & 18h, 37 °C
Sample:  Digest 20 μL
Chromato:  Shandon ODS Hypersil, 5 μm
Separation:
    Solvent A: Water (contains 0.1 % TFA);
    Solvent B: Acetonitrile : H2O = 70 : 30 (contains 0.1 % TFA)
Gradient: 20 min. linearly 0 ~ 100 % B
Flow: 1 mL/min;  Detection:  215 nm
[References]
    (1) Noreau, J. and Drapeau, G.R.: J. Bacteriol., 140, 911 (1979).
    (2) Drapeau, G.R.: Can. J. Biol. Chem., 225, 839 (1980).;
    (3) Laemmli, U.K.: Nature, 227, 680 (1970).

Endoproteinase Glu-C, Sequence grade
(Wako Catalog No. 050-05941 (50μg))
  • Origin: Staphylococus aureus V8
  • Preparation: Lyophilized, salt-free
  • Specificity:
        In Ammonium carbonate buffer (pH 7.8) or ammonium acetate buffer (pH 4.0):
            Cleaves C-terminal peptide bonds of glutamic acid.
        In phosphate buffer (pH 7.8): Cleaves C-terminal peptide bonds of glutamic acid and aspartic acid.
  • Specific activity: Indicated on each label (at 37°C with azocoll as substrate).
  • Purity: min. 90% (SDS-PAGE)
  • Performance: Cleavage after 1hr: min. 90% (with glucagon as substrate).
product
Sample: Glu C 20 μg
Chromato: Aquapore RP 300, 7 μm, 4.6 φ x 100 mm
Separation:
    Solvent A: Water (contains 0.1% TFA);
    Solbent B: Acetonitrile : H2O = 70 : 30 (contains 0.1% TFA)
      Gradient: 30 min. linear 0 - 100% B;
Flow: 0.5 mL/min;
Detection: 215 nm
product
1: Pre (1) - Glu; 2: Ala (14)-Glu (21); 3. Arg (22)- Ala (30)
Digest: 100 μg Insulin Box + 10 μg Endoproteinase Glu C
        Sequencing grade in 100 μL Ammonium Carbonate
        Buffer (25 mmol/L, pH 7.8) 1h, 25°C
Sample: 10 μL of (Digest 10 μL + Ammonium Carbonate 90 μL)
Chromato: Aquapore RP 300, 7 μm, 4.6 φ x 100mm.
Separation: Acetonitrile : H2O (contains 0.1 % TFA);
Flow: 1mL/min;
Detection: 215 nm
[References]
    (1) Houmard, J. and Drapeau, G.R.: Proc. Natl. Acad. Sci. USA.,69, 3506 (1972).;
    (2) Drapeau, G.R.: J. Biochem., 56, 534 (1978).;
    (3) Seetharama, R. and Seetharama, A.: J. Cell. Biochem., 30, 87 (1986).