Activity-based protein profiling of infected plants

Methods Mol Biol. 2012;835:47-59. doi: 10.1007/978-1-61779-501-5_3.

Abstract

Activity-based protein profiling (ABPP) is a powerful analytical method to detect and compare the activity of proteins in proteomes. This is achieved using specific activity-based probes that are often derived from inhibitors and are linked to reporter groups like rhodamine or biotin for fluorescence detection and/or affinity purification, respectively. The probes react with the active site residue of proteins and become covalently and irreversibly attached, facilitating the separation, detection and identification of the labelled proteins. In this protocol we describe all the steps required for labelling, purification and identification of labelled proteins from gels and show how activities in two proteomes can be compared. The identification of serine hydrolases from Arabidopsis plants infected with Botrytis cinerea using the trifunctional probe TriFP is used as an example.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels / chemistry
  • Affinity Labels / isolation & purification
  • Affinity Labels / metabolism*
  • Arabidopsis / metabolism
  • Arabidopsis / microbiology*
  • Botrytis / physiology*
  • Electrophoresis / methods
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / isolation & purification
  • Fluorescent Dyes / metabolism
  • Host-Pathogen Interactions*
  • Plant Proteins / chemistry
  • Plant Proteins / isolation & purification
  • Plant Proteins / metabolism*
  • Proteomics / methods*
  • Serine Proteases / chemistry
  • Serine Proteases / isolation & purification
  • Serine Proteases / metabolism

Substances

  • Affinity Labels
  • Fluorescent Dyes
  • Plant Proteins
  • Serine Proteases