From Experiment to Data Analysis: A Step-by-Step Guide to Conducting ABPP Experiments
Unlike proteomics methods,ABPP（Activity-Based Protein Profiling）not only can it identify protein expression, but it can also capture their 'active states,' making it especially suitable forcomplex regulatory functional proteins like proteases, phosphatases, deacetylases.So, how do wesystematically conduct a complete ABPP experiment? From probe selection to mass spectrometry analysis, from sample processing to data interpretation, this article will guide you step-by-step through the complete process of an ABPP experiment, providing a clear and efficient operational guide for your research.
1. Complete Process of ABPP Experiment
1. Determine Research Goals and Enzyme Families
The first step in conducting an ABPP experiment is to clarify the research goals: which type of enzymes do you want to analyze? In which biological model or under what conditions? Common goals include:
(1) Screening for proteases with abnormal activity in tumors
(2) Validating the effects of candidate drugs on target enzymes
(3) Analyzing regulatory patterns of active enzymes in inflammation or apoptosis signaling pathways
Different enzymes require the design or selection ofspecific ABPs (Activity-Based Probes), thus determining the target enzyme is crucial for subsequent probe strategy and data analysis paths.
2. Select or Customize Activity-Based Probes (ABPs)
ABPs are the key tools for successful ABPP, typically composed of the following three parts:
| Part | Function | Description |
| Warhead (Reactive Group) | Covalently binds to the enzyme's active site | such as fluorophosphonate (serine hydrolases), epoxide (cysteine proteases), etc. |
| Linker | Enhances structural flexibility and reduces steric hindrance | commonly short alkyl or PEG chains |
| Reporter | Allows detectability | biotin (for enrichment), fluorescent groups (for imaging), or alkyne/azide groups (for click chemistry) |
3. Probe Labeling of ABPP Experimental Samples
The execution steps of labeling experiments are mainly divided into two modes:
(1) Cell Lysate Labeling (in vitro)
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Extract cell/tissue proteins while maintaining enzyme activity
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Incubate with probe (30–60 min)
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Terminate the reaction and proceed with downstream processing
Suitable for enzyme screening or drug mechanism of action studies.
（2）Live Cell or Tissue Labeling (in situ/in vivo)
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Add probe directly to live cells
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Quickly lyse after the reaction is complete
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Capture the enzyme activity profile in situ
Closer to physiological conditions, but probe permeability and toxicity should be noted.
4. Enrichment and Digestion
After labeling, the next step is to enrich the labeled 'active enzymes' from the complex background:
(1) If using biotin labeling → use streptavidin magnetic beads for affinity purification
(2) If using click groups → perform click reaction and connect to enrichment carriers (e.g., Biotin-Azide)
After enrichment, perform standard proteolytic digestion reactions with trypsin, etc., to prepare peptides for subsequent mass spectrometry detection.
5. Mass Spectrometry Detection (LC-MS/MS)
Once the sample is prepared, proceed to high-resolution mass spectrometry analysis. The following strategies are recommended:
（1）Instrument Platform: such as Orbitrap Exploris 480, suitable for high-throughput, high-precision needs
（2）Separation Method: Nano-scale reversed-phase chromatography (nanoLC) enhances peptide separation capability
（3）Detection Mode: Choose between DDA (Data-Dependent Acquisition) or DIA (Data-Independent Acquisition)
（4）Quantification Method: Supports various quantification strategies such as Label-Free, TMT, iTRAQ, etc.
The ABPP experiment's raw data obtained at this stage provides core information for subsequent construction of the activity map.
6. Data Analysis and Activity Map Plotting
The ultimate goal of ABPP is to generate anactivity protein map. The data analysis process mainly includes:
（1）Peptide Identification and Quantification
Use professional software (such as MaxQuant, Proteome Discoverer) for peptide identification and quantification.
（2）Specificity Screening
Only retain proteins that have been successfully labeled (captured by the probe) and remove background signals.
（3）Activity Change Analysis
Compare the differences in enzyme activity levels between different treatment groups (e.g., drug treatment vs. control), commonly using log2 fold change + FDR filtering.
（4）Functional Annotation and Pathway Enrichment
Integrate GO, KEGG, Reactome databases for functional classification and pathway analysis of highly active or differential enzymes to extract biological significance.
（5）Map Display
Output protease activity heatmaps, molecular interaction networks, pathway enrichment maps, etc., to assist in writing papers or applying for patents.
II. Frequently Asked Questions (FAQ)
Q1: What types of proteins can ABPP experiments be used for?
Mainly used for enzyme proteins with active sites that can be covalently modified by probes, such as proteases, phosphatases, ubiquitinating enzymes, etc.
Q2: How to solve probe non-specificity?
Non-specificity can be addressed by using blank groups and competitive inhibition groups for background removal, and optimizing probe concentration and reaction time.
Q3: How to ensure data reproducibility?
It is recommended to perform ≥3 biological replicates and use methods like TMT to reduce batch effects.
III. BGI Tech: One-Stop Service Platform for ABPP Full Process
To lower the technical threshold of ABPP experiments and improve the success rate,BGI Techoffers the following services:
1. Screening and Synthesis of Various Activity Probes(Supports Customized Design)
2、Full Process Experimental Operation(From Sample Processing to Enrichment and Digestion)
3、High-Resolution Mass Spectrometry Detection and Analysis
4、Differential Protein Activity Analysis, Pathway Annotation, and Map Output
With the help of the advanced Orbitrap platform and functional proteomics analysis system, BGI Tech assists you in efficiently completing scientific exploration from activity identification to target mining.
The value of ABPP lies in its ability to not only reveal whether a protein is 'present or absent,' but also to answer 'who is alive and actively functioning.' Mastering ABPP is not just mastering a technique, but opening the door to understanding protein functional states and precise target discovery. Feel free to contactBGI Techto receive professional training and project support on ABPP workflows, helping you to transition from transcriptomics to functional genomics!
BGI Tech -- A high-quality service provider for bioproduct characterization, multi-omics mass spectrometry detection
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