A broad-spectrum fluorescence-based peptide library for the rapid identification of protease substrates.
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.
Thomas
Daniel A
DA
Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds, UK.
Francis
Peter
P
Smith
Carla
C
Ratcliffe
Steven
S
Ede
Nicholas J
NJ
Kay
Corinne
C
Wayne
Gareth
G
Martin
Steve L
SL
Moore
Keith
K
Amour
Augustin
A
Hooper
Nigel M
NM
D002154:Calpain / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006868:Hydrolysis; D020782:Matrix Metalloproteinases / Q000502:physiology; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D019151:Peptide Library; D021122:Protein Subunits / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity
D019151:Peptide Library; D021122:Protein Subunits; D010447:Peptide Hydrolases; D002154:Calpain; D020782:Matrix Metalloproteinases