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Isolation of specific tryptic peptides using chip-based bioaffinity reactor
Authors: Korecká Lucie | Mísařová Blanka | Slováková Marcela | Minc Nicolas | Viovy jean-Louis | Bílková Zuzana
Year: 2005
Type of publication: ostatní - přednáška nebo poster
Name of source: 2nd IPSO Congress Proteomics and Genomics
Publisher name: Italian Proteome Society
Place:
Page from-to:
Titles:
Language Name Abstract Keywords
cze Izolace specifických tryptických peptidů. Izolace specifických tryptických peptidů. micro-chip, anhydrotrypsin, izolace, tryptické peptidy
eng Isolation of specific tryptic peptides using chip-based bioaffinity reactor Anhydrotrypsin is bioaffinity ligand for highly specific trapping of peptides which correspond to the products generated by the action of various trypsin-like proteases and binds, in slightly acidic conditions, only specific peptides containing arginine and lysine residues at their C-termini 1,2. Anhydrotrypsin is catalytically inactive derivative of bovine trypsin in which serine residue (Ser-195) is first modified with phenylmethanesulfonyl fluoride (PMSF), an active site-directed reagent, and then the modified protein is treated with alkali results in elimination of the modified group and the serine is converted to a dehydroalanine 3. Biospecific carrier prepared by immobilization of anhydrotrypsin on magnetic or non-magnetic micro- and nanoparticles could be used for purification, isolation and preconcentration from complex mixture of tryptic peptides in one step. One of the modern trends in biochemistry is focused on the application of functional spherical nanoparticles for isolation or enzyme modification of target protein directly in a channel of microfluidic device. IMER with immobilized trypsin in tandem with chip-based IMAR with immobilized anhydrotrypsin was used for simplification of peptide mapping including the digestion of protein coupling with purification and preconcentration of peptides for following analysis by HPLC or MS. High purity and preconcentration of peptides isolated on immobilized anhydrotrypsin significantly facilitated the identification of protein fingerprint even for protein in submicromolar concentration. micro-chip, anhydrotrypsin, isolation, tryptic peptides