Activity‐Based Protein Profiling


Activity‐based protein profiling is a method to study a subset of the enzymatically active proteome. This method uses chemical probes that covalently react with active enzymes. These labelled proteins can subsequently be analysed by means of a detection tag on the probe. A diverse set of probes has been developed for many enzyme classes, such as serine hydrolases, proteases, glycosidases and kinases. Different analytical techniques are currently available to visualise, identify and quantify probe‐labelled proteins with high efficiency. Activity‐based protein profiling has well‐developed applications in discovering new drug targets and in profiling inhibitors for potency and selectivity. Activity‐based protein profiling will, therefore, continue to aid research both in fundamental biology and drug discovery.

Key Concepts

  • Activity‐based protein profiling (ABPP) is a method to study an enzymatically active subset of the proteome and complements abundance‐based proteomics.
  • An activity‐based probe is a compound that covalently labels only the active form of an enzyme.
  • Activity‐based probes have been developed for several classes of enzymes, such as serine hydrolases, proteases, lipases and kinases.
  • Every ABPP experiment consists of two parts: an activity‐dependent labelling part, followed by an analytical part to visualise and characterise this labelling event.
  • Different analytical platforms have been developed for ABPP, including SDS‐PAGE, LC‐MS, CE‐LIF, FluoPol, EnPlex and microarray platforms.
  • ABPP is mainly used for target discovery by comparing different proteomes (comparative ABPP) and for characterising inhibitor selectivity and potency (competitive ABPP).
  • ABPP is a powerful method for drug discovery.
  • It is surprising that ABPP is not more widely used outside of specialised labs developing new probes.

Keywords: activity‐based protein profiling; proteomics; activity‐based probe; enzymes; drug discovery

Figure 1. Labelling enzymes with an activity‐based probe. (a) General activity‐based probe design, with fluorophosphonate‐biotin as example. (b) Probe labelling cartoon: two‐step labelling using bioorthogonal chemistry (BOC) is optional for probes equipped with a suitable tag. (c) Mechanism of serine hydrolase labelling: catalytic triad reacting with the fluorophosphonate trap.
Figure 2. Visualisation of ABPP analytical platforms: SDS‐PAGE, CE‐LIF, LC‐MS, microarray and FluoPol.
Figure 3. ABPP experiments. (a) Comparative ABPP. (b) Competitive ABPP.


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van Rooden, Eva J, Bakker, Alexander T, Overkleeft, Herman S, and van der Stelt, Mario(Jan 2018) Activity‐Based Protein Profiling. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0023406]