Lysyl endopeptidase is a serine protease that cleaves peptide bonds at the carboxy-terminal side of lysine residues with a high degree of specificity. It is inhibited by TLCK, PMSF, and DFP but not by TACK. Taking advantage of its excellent specificity, lysyl endopeptidase is used for peptide fragmentation and peptide mapping for analysis of the primary structure of proteins.
It is originally isolated from a soil bacterium by Masaki et al., cleaves specifically the peptides on the carboxyterminal side of Lysine residues. This enzyme is very useful both in protein sequence analysis and in enzymatic synthesis of Lys-X compounds.
-Hydrolysable substrates???Tos-Lys-OMe, Bz-Lys-NH2, Bz-Lys-pNA, Lys-pNA
-Unhydrolysable substrates???Bz-Arg-NH2, Bz-Arg-pNA, Arg-pNA
Comparison of In-gel Digestion using Trypsin (Tp), Lysl Endopeptidase (Lep) and Lep Combined with Tp (Lep + Tp)
BSA band (100 ng) resolved by SDS-PAGE was in-gel digested with Tp, Lep and Lep + Tp and analyzed by MALDI-TOFMAS. The figure shows the individual mass spectra. The evaluation of these peptidases is summarized in the table.
Mass spectrum using Tp and Tp + Lep
When Tp is used concurrently with Lep, additional peaks were found around m/z 2000 compared to when only Tp is used. It shows that the recovery rate of peptide increases.
Table. Comparison ofTp, Lep, and Tp + Lep
?The coverage (percentage of available peptides in the entire sequence) obtained by database search using a parameter setting of Missed Cleavage = 0 was subtracted from the coverage obtained by using a parameter setting of Missed Cleavage = 1.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.
These results indicate there are very few missed cleavages obtained by Lep digestion. When Tp is used concurrently with Lep, missed cleavages decrease and the number of identified peptides increase compared to when only Tp is used.
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