Activity-Based Protein Profiling Experimental Process and Key Operation Points

The Activity-Based Protein Profiling (ABPP) experimental process involves several critical steps from probe design, sample processing, to signal detection and data analysis. The following isthe standard ABPP experimental procedure and key points for each step, applicable to most enzyme studies (such as serine hydrolases, proteases, deacetylases, etc.).

 

I. Overview of the ABPP Experimental Process

1. Preparation of the Activity Probe

2、Sample Preparation (cells, tissues, or purified enzymes)

3、Probe Labeling (incubation)

4、Enrichment or Separation (optional)

5、Analysis and Detection (SDS-PAGE/Western blot or LC-MS/MS)

6、Data Analysis (qualitative or quantitative)

 

II. Detailed Explanation and Key Points for Each Step

1. Activity Probe Design and Preparation

(1) Structural Composition:

• Warhead: Choose a reactive group that covalently binds to the active site of the target enzyme (such as fluorophosphonate, chloromethyl ketone, etc.).

• Linker: Can modulate spatial conformation to avoid interference with enzyme recognition.

• Reporter Tag: Can be a fluorescent group, biotin, radioactive label, or 'click chemistry' handle (such as alkyne).

(2) Key Points:

• Design the reactive group based on the catalytic mechanism of the target enzyme.

• For LC-MS usage, it is recommended to use small molecule tags or 'click' probes for easier subsequent purification and detection.

 

2. Sample Preparation

(1) Types:

Cell lysates, tissue homogenates, serum, purified enzymes, live cells, animal tissues, etc.

(2) Key Points:

• Maintain enzyme activity: Buffer solutions (commonly PBS, Tris-HCl) should be included in lysates, avoid strong denaturants and high concentrations of detergents.

• Pre-cleaning: Remove background interfering proteins.

• Maintain cold chain: Prepare using low temperatures (0–4°C), add protease inhibitors (avoid inhibitors of target enzymes).

 

3. Probe Labeling (Incubation)

(1) Condition Optimization:

• Probe concentration: Typically in the range of 0.1–10 μM.

• Time: 10–60 minutes.

• Temperature: Usually 37°C, some cell experiments can be conducted at room temperature or 4°C to reduce non-specific labeling.

(2) Key Points:

• Control groups can be set: Such as untreated, heat-inactivated samples, or samples with known enzyme inhibitors added.

• Competitive ABPP: Add small molecule inhibitors to pre-treat samples and test if they can competitively block probe binding.

 

4. Enrichment (Optional)

Applicable to non-fluorescent tags, such as probes with biotin/alkyne, requiring click reaction and affinity purification.

(1) Procedure:

• Click Chemistry: alkyne + azide-Cy5/biotin, click under Cu(I) catalysis.

• Enrichment Method: Streptavidin magnetic beads pull-down (biotin-labeled probe).

(2) Key Points:

• Reaction buffer should have good redox control.

• When eluting samples with magnetic beads, care should be taken to avoid non-specific binding.

 

5. Analysis and Detection

  

Common Methods	Description
SDS-PAGE + Fluorescence Scanning	Suitable for fluorescent-tagged probes; fast and intuitive.
Western Blot	If the probe is biotin-labeled, detection with HRP- or fluorescence-labeled streptavidin is required.
LC-MS/MS	Combined with affinity purification, qualitative and quantitative analysis of enzyme activity profiles; high throughput but time-consuming.

 

Key Points:

(1) Protein quantification should be accurate to ensure consistent sample loading across groups.

(2) Mass spectrometry samples should be subjected to enzymatic digestion, desalting, and purification before analysis.

 

6. Data Analysis

Based on the type of experiment:

(1) Qualitative: Enzyme type identification, protein identification rate (e.g., ABPP-MS).

（2）Quantitative: Assess changes in enzyme activity using isotope labeling methods (SILAC, TMT) or label-free approaches (LFQ).

（3）Image Analysis: Fluorescent gels can be analyzed for band intensity using ImageJ.

 

III. Summary of Key Operational Precautions

  

Project	Key Points
Control Group Setup	Blank groups, competitive groups, and inhibitor groups must be set to ensure specificity.
Maintaining Enzyme Activity	Avoid extreme pH, denaturants, and heat treatment throughout the sample processing.
Probe Optimization	Choose the right warhead and tag; probes cannot be universally applied to different enzyme classes.
Reducing Non-specificity	Use appropriate blocking agents, add competitive substrates, and control temperature.

 

Activity-Based Protein Profiling (ABPP) combines chemical probes with proteomics to specifically detect active enzymes in complex biological samples, revealing their functional status and regulatory mechanisms. By designing effective activity probes, optimizing sample treatment and labeling conditions, and integrating efficient detection methods (such as fluorescence imaging or mass spectrometry analysis), ABPP can be used for enzyme function research and drug target identification, showing great potential in disease mechanism exploration and clinical biomarker discovery. Mastering its experimental processes and key operational points will provide a solid foundation for advancing functional proteomics research.

 

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