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Chip-based magnetic separation technique with immobilized trypsin and anhydrotrypsin for isolation of specific peptides of neurotensin

Rok: 2005

Druh publikace: článek ve sborníku

Název zdroje: 11th International Symposium on Separation Science (ISSS 2005)

Název nakladatele: Univerzita Pardubice

Místo vydání: pardubice

Strana od-do: 234

Tituly:

Jazyk	Název	Abstrakt	Klíčová slova
cze	Separace založená na biočipové technologii, specifická izolace tryptických peptidů pomocí anhydrotrypsinu.	Separace založená na biočipové technologii, specifická izolace tryptických peptidů pomocí anhydrotrypsinu.	micro-chip, magnetické nosiče, izolace, neurotensin
eng	Chip-based magnetic separation technique with immobilized trypsin and anhydrotrypsin for isolation of specific peptides of neurotensin	The isolation of proteins and peptides in the area of biosciences and biotechnology is now the subject of interest. Magnetic separation techniques have several advantages in comparison with standard separation procedures. Functional spherical nanoparticles with controlled tendency to aggregate and controlled level of nonspecific sorption could be focused in channel of magnetically active microfluidic device for isolation or enzyme modification of target protein directly in a microchannel. Biospecific ligand anhydrotrypsin enables 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]. 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 [2]. Magnetic affinity reactor prepared by immobilization of anhydrotrypsin on magnetic nanoparticles was used for purification, isolation and preconcentration of specific peptides from complex mixture of tryptic peptides in one step. IMER (immobilized magnetic enzyme reactor) with trypsin in tandem with chip-based IMAR (immobilized magnetic affinity reactor) with anhydrotrypsin were used for simplification of peptide mapping including the digestion of protein coupling with purification and preconcentration of tryptic peptides of neurotensin for following analysis by HPLC and 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, magnetic carrier, isolation, neurotensin

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