DIGE-ABPP by click chemistry: pairwise comparison of serine hydrolase activities from the apoplast of infected plants

Methods Mol Biol. 2014;1127:183-94. doi: 10.1007/978-1-62703-986-4_15.

Abstract

Activity-based protein profiling (ABPP) is a targeted functional proteomics method that displays the active proteome by using small molecule probes that react covalently with the active sites of protein classes. Comparison of activity profiles from two different samples is not always easy, especially when using probes that generate too many signals. For accurate comparison of protein activities between two proteomes, we developed difference gel electrophoresis ABPP (DIGE-ABPP), which compares two fluorescently labeled proteomes in the same gel lane. This protocol describes the labeling of two proteomes with alkyne-labeled probes, followed by the coupling with two different fluorophores using "click chemistry," the separation of mixed proteomes on protein gels, and the quantification and comparison of the activity profiles. We applied DIGE-ABPP to investigate differential serine hydrolases activities in the apoplast of Nicotiana benthamiana challenged with Pseudomonas syringae p.v. tomato DC3000.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetone / chemistry
  • Chemical Precipitation
  • Click Chemistry / methods*
  • Electrophoresis, Gel, Two-Dimensional / methods*
  • Fluorescence
  • Molecular Probes / metabolism
  • Proteome / metabolism
  • Proteomics / methods*
  • Pseudomonas syringae / physiology*
  • Serine Proteases / metabolism*
  • Tobacco / cytology*
  • Tobacco / enzymology
  • Tobacco / microbiology*

Substances

  • Molecular Probes
  • Proteome
  • Acetone
  • Serine Proteases