Chemically modified, immobilized trypsin reactor with improved digestion efficiency

J.R. Freije, P.P. Mulder, W. Werkman, L. Rieux, H.A G Niederlander, Sabeth Verpoorte, Rainer Bischoff*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

70 Citations (Scopus)

Abstract

Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K-M and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.

Original languageEnglish
Pages (from-to)1805-1813
Number of pages9
JournalJournal of Proteome Research
Volume4
Issue number5
DOIs
Publication statusPublished - 2005

Keywords

  • immobilized trypsin
  • digestion reactors
  • chemical modification
  • acetylation
  • proteomics
  • PROTEIN IDENTIFICATION
  • MASS-SPECTROMETRY
  • SEQUENCE DATABASES
  • CHROMATOGRAPHY
  • STABILIZATION
  • PROTEOMICS
  • SYSTEM
  • ONLINE
  • CHIP
  • ELECTROPHORESIS

Cite this