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Activity-Based Probe Experimental Design

 

Activity-based probes (ABPs) directly visualize and measure metabolite and enzyme interactions using modular chemical probes that can be designed for microscopy or fluorescence-activated cell sorting (FACS) and for physical enrichment and detection by mass spectrometry. Substrate-mimicking photoaffinity-based probes are used to capture protein-metabolite interactions, especially in cases where a true activity-based probe has not been developed. These probes are particularly useful for studying non-enzymatic protein-metabolite interactions, such as those involving transport proteins.

  

Access the workflow steps for each specific activity-based probe experimental design process.

activity-based probes
Activity-based probes (ABPs) are substrate-based, small organic compounds that enable the capture of the functional state of proteins in vivo and in vitro, isolation of cells with targeted functions, elucidation of biochemical pathways, and annotation of proteins of unknown function. Using advanced analytical tools, researchers gain spatio-temporal understanding of functional proteins in biochemical processes. (Photo by Andrea Starr | Pacific Northwest National Laboratory)

ABPs capture the interactions of small molecules with proteins using various analytical tools, providing information on movement, localization, and the functional state of proteins/enzymes. The current ABP-imaging pipeline uses fluorescence microscopy, FACS, and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 

three panels of probe images of Yarrowia lipolytica
Activity-based probes can be used to image protein activity such as enzymes or transporters by microscopy. In one example, Yarrowia lipolytica mannose membrane transporters are marked by a mannose substrate-mimicking photoaffinity-based probe linked to a fluorophore. (Image by Erin Bredeweg | Environmental Molecular Sciences Laboratory)
1. Probe Development 
  • Design synthetically tractable probes using known substrate and biochemical information of the targets
  • Synthesize, isolate, and characterize probes (nuclear magnetic resonance and mass spectrometry)
  • Validate probes with known protein targets
  • Choose one with desired stability, solubility, and selectivity
  • Probes can be redesigned based on the validation to obtain desired selectivity
  • Developed probes can be used from step 2 for multiple users’ systems 
2. ABP Labeling of Samples 
  • Use the dose response (~1 hr) to select the lowest working probe concentration in the nonreactive buffer 
  • Expose photoaffinity (UV-activatable) probes to UV light for ~7 min 
  • Attach a fluorophore to the probe after cell incubation/fixation via click chemistry (1 hr) 
3. ABP-Sample Imaging/Sorting 
  • Wash out the remaining probe and fluorophore
  • Image live or fixed cells with fluorescence confocal microscopy (Airyscan) 
  • Include a no-probe control (DMSO) for background 
  • Assess by a competition assay with the true substrate 
  • Live/fixed cells can be sorted by FACS after probe labeling (influx) 

capture the interactions of small molecules with proteins using liquid chromatography–mass spectrometry (LC-MS) proteomics. This approach provides information on protein identity, relative abundance, and the functional state of proteins or enzymes.

1. Probe Development 
  • Design synthetically tractable probes using known substrate and biochemical information of the targets
  • Synthesize, isolate, and characterize probes (NMR and mass spectrometry)
  • Validate probes with known protein targets
  • Choose one with desired stability, solubility, and selectivity
  • Probes can be redesigned based on the validation to obtain desired selectivity
  • Developed probes can be used from step 2 for multiple users’ systems 
2. ABP Labeling of Samples 
  • Use 1–2 mg/mL protein samples for probe labeling 
  • Use probes on intact cells or lysate; lyse whole cells; determine protein concentration
  • Use the dose response to select the lowest working probe concentration in the nonreactive buffer (SDS-PAGE)
  • For photoaffinity (UV-activatable) probes, expose to UV light 
  • Attach a pull-down moiety (e.g., biotin) to the probe after cell incubation or fixation via click chemistry 
  • Generate competitively labeled samples by pretreating with the true substrate  
3. ABP-Sample Proteomics 
  • Enrich probe labeled proteins using streptavidin beads
  • Use a no-probe control for background binding 
  • Perform tryptic (12 hrs) digestion to release peptides from streptavidin beads
  • Send peptides for LCMS analysis
4. Data Processing 
  • Proteomics processing 
  • Background subtraction for quantitation
  • ABP enriched protein identification
Contact

Sankar Krishnamoorthy, sankarganesh.krishnamoorthy@pnnl.gov