Lysyl Endopeptidase®, Mass Spectrometry Grade (Lys-C)
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 (Lys-C) is a freeze dried product that retained sufficient activity for in-gel digestion and packed in very small quantities for convenience purposes.
- High specificity and efficiency of protein digestion allow for easy database searches by peptide mass.
- Improved cleavage at lysine residue and increase in the number of peptides are obtained by combination with trypsin.
- 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||Tp + Lep|
|Cleavage site||C terminal of Arg + Lys||C terminal of Lys||C terminal of Arg + Lys|
|Missed cleavage (Rates of missed cleavage)※||Many (8%)||Very few (0%)||Few (3%)|
|No. of identified peptides||17||19||22|
Data provided by: Wada, Y., Osaka Women’s and Children’s Hospital
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.
Mass spectrum: Comparison of Trypsin (Tp), Lysyl Endopeptidase® (Lep) + Tp
- Wada, Y. and Kadoya, M.: J. Mass Spectrom., 38, 117(2003).
- Shevchenko, A., Wilm, M., Vorm, O. and Mann., M.: Anal. Chem., 68, 850(1996).
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