Activity-Based Protein Profiling (ABPP) Service for Small-Molecule Target Discovery

Activity-based protein profiling is a chemical proteomics strategy that links small molecules to their functional protein targets in complex biological systems. By using activity-based probes rather than expression-only readouts, ABPP reveals which proteins are truly engaged and active under specific conditions. At Creative Proteomics, ABPP is applied to support small-molecule target identification, enzyme activity mapping, and mechanism-of-action studies in a range of biological models.

ABPP service highlights

Function-centric proteomics that focuses on catalytically active proteins, not just expression levels
Small-molecule target deconvolution and off-target discovery in native-like environments
Flexible lysate, live-cell, and tissue formats aligned to your biological model
LC–MS/MS–based workflows with transparent quantification and pathway interpretation

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What Is Activity-Based Protein Profiling?

Activity-based protein profiling (ABPP) combines chemistry and proteomics. Activity-based probes react covalently with active sites of specific enzyme classes or with proteins bound by a modified small molecule. Labeled proteins are then enriched and analyzed by mass spectrometry to generate a global "activity map" under defined conditions.

Unlike expression-focused proteomics, ABPP enriches only proteins that are catalytically competent or directly engaged by a probe-derived small molecule. This activity-centric view is the foundation for downstream target discovery and mechanism-of-action studies.

Structure of an activity-based probe (ABP)

An ABP typically includes three functional elements:

Reactive group (warhead)
Covalently engages the enzyme active site or a defined binding pocket after recognition. Common warheads are chosen based on the enzyme class or the small molecule scaffold.
Linker region
Provides spacing and flexibility between the reactive group and the reporter handle. It helps maintain the binding properties of the small molecule and reduces steric hindrance.
Reporter or clickable handle
Enables downstream detection or enrichment. This may be a fluorophore, a biotin tag, or a small alkyne/azide group that later undergoes click chemistry to add biotin or other reporter tags.

In vitro, cellular, and in vivo labeling modes

ABPP experiments can be configured in multiple formats depending on the biological question:

Lysate-based ABPP (in vitro) for enzyme family screening and mechanistic profiling
Live-cell ABPP (in situ) for mapping protein activity in near-physiological contexts
Tissue or in vivo ABPP for capturing activity states and drug–target interactions in animal models or ex vivo tissues

These flexible formats allow ABPP to move from reductionist biochemical systems to complex tissues and in vivo models while preserving an activity-focused readout.

Technical Services

Experimental Modes Service Scope Method Comparison Workflow Platform Sample Guide Deliverables FAQ Get a Custom Proposal

ABPP for Drug Discovery and Mechanism-of-Action Studies

In drug discovery and development, ABPP helps address practical questions about which proteins are active, which are engaged by a compound, and how these events connect to phenotypes.

Moving from protein expression to functional activity

Conventional proteomics measures protein abundance, but many drug targets are regulated at the level of activity, not expression. ABPP focuses on catalytically active proteins, capturing only those enzymes that are "on" and able to react with the probe.

This distinction is critical when prioritizing drug targets or interpreting phenotypes, because changes in expression do not always correspond to changes in functional enzyme activity.

Linking small molecules to direct protein targets

ABPP uses tailored probes derived from active small molecules or enzyme family–selective scaffolds. These probes form covalent bonds with target proteins, allowing unambiguous enrichment and identification by LC–MS/MS. This helps clarify primary targets, off-targets, and pathway context in one experiment.

For hit validation and lead optimization, this direct link between a small molecule and its protein binding partners provides a mechanistic basis for observed pharmacology.

Typical use cases

Identification of protein targets for bioactive small molecules
Characterization of enzyme families (proteases, phosphatases, dehydrogenases, deacetylases, and more)
Comparative activity profiling across disease models, treatments, or genetic backgrounds
Supporting hit-to-lead optimization with target engagement and selectivity data

ABPP Experimental Modes and Study Designs

Lysate-based ABPP

Ideal for broad profiling of enzyme activity across conditions or treatment arms. This format simplifies probe delivery and allows tight control over reaction conditions.

Live-cell ABPP

Suitable for studying enzyme activity and small-molecule engagement in intact cells. This format captures protein activity in a more physiological context, while accounting for cell permeability and intracellular localization.

Tissue and in vivo ABPP

Designed for animal or tissue-based models where preserving native architecture and microenvironments is important. Sample preparation is tailored to maintain crosslinks and minimize activity loss.

Competitive ABPP for target validation

Used to confirm specific binding of a lead molecule to its targets:

Pre-incubate samples with the unlabeled small molecule.
Introduce a broad-spectrum or class-specific ABP.
Observe reduced labeling at proteins where the small molecule occupies the active site, indicating target engagement.

Visualization and localization of drug–target engagement

Fluorescent or imaging-compatible probes can be used to visualize cells or tissues, allowing qualitative assessment of probe uptake and target engagement patterns.

Custom assay and analysis configurations

Different projects may require:

Focused panels of candidate targets
Global, unbiased proteome-wide profiling
Tailored quantification strategies for subtle activity changes
Integration with orthogonal assays such as biochemical kinetics or binding studies

ABPP Solutions: What We Offer at Creative Proteomics

Creative Proteomics provides a portfolio of ABPP solutions that can be tailored to specific discovery and development needs.

Small-Molecule Target Identification and Deconvolution

Convert your active compound into an ABP where needed.
Capture direct targets and key off-targets in relevant models.
Provide a ranked target list with concise functional notes.

Enzyme Family Activity Mapping

Use family-selective probes to profile active proteases, phosphatases, dehydrogenases, deacetylases, and more.
Compare activity patterns across cell lines, tissues, or conditions.
Deliver quantitative activity signatures for target and pathway selection.

Drug and Perturbation Response ABPP

Run treated vs control ABPP to track activity shifts and target engagement.
Highlight enzymes that are activated, inhibited, or competitively blocked.
Support MoA refinement and lead optimization with comparative readouts.

Metabolism and Signaling Pathway Activity Profiling

Focus ABPP on metabolic and signaling nodes of interest.
Integrate results with pathway resources to reveal impacted routes.
Summarize pathway-level effects linked to efficacy or toxicity.

Tissue and Subcellular Activity Mapping

Apply ABPP in tissues, primary cells, or fractions such as mitochondria or membranes.
Map where targets are functionally active under different conditions.
Provide spatially informed activity profiles for target and biomarker strategy

Integrated Target Validation Packages

Combine ABPP with orthogonal binding and activity assays.
Confirm on-target engagement and assess selectivity within related families.
Deliver a compact data package to support internal decision making.

Choosing Between ABPP and Related Target/Activity Profiling Technologies

Comparison point	ABPP	Affinity pull-down (biotin / resin)	Thermal shift / target engagement (TSA, CETSA, TPP, DSC-like)	DARTS (Drug Affinity Responsive Target Stability)	Standard quantitative proteomics
Primary readout	Covalent labeling of catalytically active enzymes or probe-bound proteins	Physical binding of proteins to a tagged or immobilized ligand	Change in protein stability or melting behavior upon ligand binding	Protease resistance after ligand binding	Protein abundance (expression level)
Key question addressed	Which enzymes are active, and which proteins does my small molecule directly engage?	Which proteins bind to my compound under defined conditions?	Does my unmodified compound engage targets in cells/tissues, and how strongly?	Does my compound stabilize specific proteins against limited proteolysis?	Which proteins are up- or downregulated by treatment or genotype?
Sample & live-cell compatibility	Lysates, live cells, sometimes tissues; in vitro / in situ / in vivo formats	Mainly lysates; in vitro	Live cells, tissues, or lysates depending on format	Mostly lysates; sometimes cells after lysis	Lysates from cells or tissues; broad compatibility
Compound modification required?	Usually yes (ABP or warhead with clickable handle), or use class-selective ABPs	Yes (biotin tag or solid-support linker)	No, uses unmodified compound	No, uses unmodified compound	No
Key strengths	Activity-centric; distinguishes active vs inactive pools; proteome-wide target and selectivity profiling; flexible formats	Conceptually simple; good for strong binders; works for many protein classes	Direct readout of target engagement in native systems; no derivatization; scalable to proteome level	Label-free; useful as orthogonal validation for selected targets	Unbiased view of expression changes; mature, robust workflows
Main limitations	Needs suitable probe chemistry; not all targets can be covalently tagged; chemistry adds cost/lead time	Tags can alter binding; weak or transient interactions often missed; mainly lysate-based and not activity-selective	Reports stability, not catalytic activity; some binders show minimal thermal shift; requires careful optimization	Lower throughput and less quantitative at proteome scale; indirect readout; sensitive to protease conditions	Does not report activity or direct binding; cannot distinguish active vs inactive protein pools
Best suited for	Activity-centric, proteome-wide target mapping and selectivity profiling when probe chemistry is feasible	First-pass binding partner lists when ABPP probes are not yet available	Unmodified-compound target engagement in cells or tissues; orthogonal confirmation of ABPP/pulldown hits	Focused validation of a small number of candidate targets	Understanding downstream pathway and expression changes, not direct target engagement

For conformational and interaction-induced changes complementary to ABPP, consider our LiP-MS service.

Our ABPP Workflow for Small-Molecule Target Profiling

Project scoping & study design

Define biological question, model system, target classes, control groups, and comparison arms. Select lysate, live-cell, or tissue format.

Probe strategy & chemistry

Choose suitable class-selective ABPs or design a probe from your small molecule (photoaffinity and/or clickable handle) while preserving activity.

Pilot optimization

Test probe concentration, incubation time, and buffer conditions in a small panel of samples to balance sensitivity and specificity.

Sample preparation & labeling

Prepare cell or tissue lysates, live cells, or fractions. Incubate with the probe or derivatized compound under optimized conditions.

Crosslinking & click chemistry

Apply UV (for photoaffinity probes) to covalently fix probe–protein complexes, then perform click chemistry to attach biotin or other reporter tags.

Affinity enrichment & cleanup

Capture labeled proteins with streptavidin or matched matrices. Wash to remove non-specific binders and prepare material for LC–MS/MS.

Digestion & LC–MS/MS acquisition

Digest enriched proteins to peptides and run high-resolution LC–MS/MS using appropriate acquisition (DDA or DIA) and QC checks.

Data analysis & interpretation

Identify and quantify enriched proteins, compare treatment vs control, and annotate targets with pathways and functions to generate decision-ready lists.

Differential Scanning Calorimetry Instrumentation and Platform Capabilities

Nano-LC autosampler and column system: Robust peptide separation before MS.

Low carryover and precise injection for label-free or isobaric quantitation.
Stable gradients up to ~120–180 min for complex samples.

High-resolution LC–MS/MS system: Orbitrap or Q-TOF mass spectrometer coupled to nano-LC.

Resolving power: ≥60,000 at m/z 200 for confident peptide identification.
Mass accuracy: typically ≤5 ppm with internal calibration.
Scan speed: supports fast DDA/DIA for complex ABPP enrichments.
Nano-flow LC (≈200–300 nL/min) with long analytical columns (e.g., 15–50 cm, sub-2 µm particles) for deep coverage.

Orbitrap Exploris 240 (Figure from Thermo)

Thermo Orbitrap Exploris 240

EASY-nLC 1200 (Figure from Thermo)

Sample Requirements and Submission Guidelines for ABPP Services

Category	Requirements	Details / Notes
Sample types	Cell lysates, live cells, or tissue samples	Compatible with most mammalian cell lines, primary cells, and fresh/frozen tissues.
Minimum material	50–200 µg total protein per condition	Amount depends on probe chemistry and enrichment depth.
Cell number (live-cell ABPP)	1–5 × 10⁶ cells per condition	Adherent or suspension cells; avoid over-confluence for consistent probe uptake.
Lysis buffer	Mild, MS-compatible buffers	Avoid high detergents, SDS, glycerol, DTT, or components interfering with click chemistry.
Protease / phosphatase inhibitors	Recommended	Use inhibitor cocktails that do not contain azides, thiols, or copper chelators.
Sample quality	Fresh or snap-frozen; minimal freeze–thaw	Degraded or contaminated samples reduce ABPP activity detection.
Shipping conditions	Dry ice (frozen) / cold pack (cells)	Use clearly labeled tubes with sample name, condition, protein amount, and date.
Optional add-ons	Pre-lysis, protein quant, trial ABPP	We can prepare lysates, run pilot probe tests, or quantify protein before enrichment.

Deliverables: What You Get from Our ABPP Service

Typical ABPP project outputs may include:

Curated list of identified probe-enriched proteins and candidate targets
Relative activity or enrichment values across experimental groups
Volcano plots, heatmaps, and other visual summaries of activity changes
Functional and pathway annotation for significantly enriched proteins
Clear description of experimental conditions and data processing steps

In-gel ABP fluorescence with a target band competed by increasing small-molecule concentrations and equal total protein loading.

In-gel ABP fluorescence and total protein control showing dose-dependent competition of the target band.

Volcano plot of ABPP results with significantly enriched proteins such as ACAT1 and ALDH2 highlighted in purple.

Volcano plot of ABPP quantitative data, highlighting significantly enriched protein targets.

Hierarchical clustering heatmap of ABPP-derived target activity across vehicle, low-dose, and high-dose conditions.

Heatmap showing clustered, dose-dependent changes in relative target activity across conditions.

Simplified glycolytic–TCA pathway map with ABPP-enriched enzymes marked as coloured nodes of different sizes.

Pathway map integrating ABPP targets into a glycolytic–TCA network, with node colour and size encoding activity and enrichment.

You May Want to Know

What problems does ABPP actually solve in drug discovery?

ABPP helps link a phenotype or hit compound to functional, active protein targets rather than just expression changes. It is especially useful for deconvoluting on- and off-targets in enzyme families where activity is decoupled from protein level.

Is ABPP only for covalent inhibitors?

No. Classical ABPP uses covalent warheads, but non-covalent ligands can be studied via photoaffinity probes or competitive ABPP, where the unmodified compound competes with a broad-spectrum probe.

When does ABPP make more sense than a thermal shift or CETSA-style assay?

Choose ABPP when you need a proteome-wide, activity-centric map (which enzymes are “on” and engaged) and not just a stability shift. Thermal shift / CETSA are better if you mainly want to confirm global target engagement of an unmodified drug with simpler readouts.

What kind of targets are best suited for ABPP?

ABPP works best for proteins with defined active-site chemistry, such as serine hydrolases, cysteine proteases, phosphatases, deacetylases, and many metabolic enzymes. These classes already have well-developed warheads and probe chemistries.

Can ABPP be done in live cells or tissues, or only in lysates?

Yes, ABPP can run in lysates, live cells, and in vivo / tissue settings. In practice, many projects optimize conditions in lysates first, then extend to live-cell or in vivo formats once probe uptake, toxicity, and signal quality are understood.

Do I always need a custom probe for my compound?

Not always. Many projects start from family-selective probes that already exist for a given enzyme class. Custom compound-derived probes or competitive ABPP are added when you need compound-specific selectivity and MoA data.