UNIGE document Scientific Article
previous document  unige:1235  next document
add to browser collection
Title

Trypsin immobilization on three monolithic disks for on-line protein digestion

Authors
Gaud, Nicolas
Stella, Cinzia
Published in Journal of Pharmaceutical and Biomedical Analysis. 2008, vol. 48, no. 2, p. 398-407
Abstract The preparation and characterization of three trypsin-based monolithic immobilized enzyme reactors (IMERs) developed to perform rapid on-line protein digestion and peptide mass fingerprinting (PMF) are described. Trypsin (EC 3.4.21.4) was covalently immobilized on epoxy, carbonyldiimidazole (CDI) and ethylenediamine (EDA) Convective Interaction Media (CIM) monolithic disks. The amount of immobilized enzyme, determined by spectrophotometric measurements at 280nm, was comprised between 0.9 and 1.5mg per disk. Apparent kinetic parameters Km* and Vmax*, as well as apparent immobilized trypsin BAEE-units, were estimated in flow-through conditions using N-alpha-benzoyl-L-arginine ethyl ester (BAEE) as a low molecular mass substrate. The on-line digestion of five proteins (cytochrome c, myoglobin, alpha1-acid glycoprotein, ovalbumin and albumin) was evaluated by inserting the IMERs into a liquid chromatography system coupled to an electrospray ionization ion-trap mass spectrometer (LC-ESI-MS/MS) through a switching valve. Results were compared to the in-solution digestion in terms of obtained scores, number of matched queries and sequence coverages. The most efficient IMER was obtained by immobilizing trypsin on a CIM EDA disk previously derivatized with glutaraldehyde, as a spacer moiety. The proteins were recognized by the database with satisfactory sequence coverage using a digestion time of only 5min. The repeatability of the digestion (R.S.D. of 5.4% on consecutive injections of myoglobin 12microM) and the long-term stability of this IMER were satisfactory since no loss of activity was observed after 250 injections.
Keywords Amino Acid SequenceEnzymes, Immobilized/chemistryMolecular Sequence DataProteins/analysisSpectrometry, Mass, Electrospray IonizationTrypsin/chemistry
Stable URL http://archive-ouverte.unige.ch/unige:1235
Full text
Article (Author postprint) (487 Kb) - document accessible for UNIGE members only Limited access to UNIGE
Identifiers
PMID: 18242915
Structures

171 hits

1 download

Update

Deposited on : 2009-03-27

Export document
Format :
Citation style :