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A broad‐spectrum fluorescence‐based peptide library for the rapid identification of protease substrates
Author(s) -
Thomas Daniel A.,
Francis Peter,
Smith Carla,
Ratcliffe Steven,
Ede Nicholas J.,
Kay Corinne,
Wayne Gareth,
Martin Steve L.,
Moore Keith,
Amour Augustin,
Hooper Nigel M.
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500153
Subject(s) - protease , proteases , peptide , trypsin , peptide library , biochemistry , förster resonance energy transfer , tripeptide , biology , chymotrypsin , computational biology , chemistry , peptide sequence , combinatorial chemistry , enzyme , fluorescence , physics , quantum mechanics , gene
Abstract Identification of peptide substrates for proteases can be a major undertaking. To overcome issues such as feasibility and deconvolution, associated with large peptide libraries, a ‘small but smart’ generic fluorescence resonance energy transfer rapid endopeptidase profiling library (REPLi) was synthesised as a tool for rapidly identifying protease substrates. Within a tripeptide core, flanked by Gly residues, similar amino acids were paired giving rise to a relatively small library of 3375 peptides divided into 512 distinct pools each containing only 8 peptides. The REPLi was validated with trypsin, pepsin, the matrix metalloprotease (MMP)‐12 and MMP‐13 and calpains‐1 and ‐2. In the case of calpain‐2, a single iteration step involving LC‐MS, provided the definitive residue specificity from which a highly sensitive fluorogenic substrate, (FAM)‐Gly‐Gly‐Gly‐Gln‐Leu‐Tyr‐Gly‐Gly‐DPA‐Arg‐Arg‐Lys‐(TAMRA), was then designed. The thorough validation of this ‘small but smart’ peptide library with representatives from each of the four mechanistic protease classes indicates that the REPLi will be useful for the rapid identification of substrates for multiple proteases.