Target Identification Service

Target identification service help researchers for studying the interactions between small-molecule drugs, natural compounds, or other bioactive molecules and proteins. By employing chemoproteomics, mass spectrometry, and bioinformatics techniques, this service systematically elucidates the relationship between small molecules and their target proteins. The methods for target identification are broadly categorized into labeled approaches (e.g., CCCP and ABPP) and label-free ones (e.g., DARTS, CETSA, and SPROX). These methods are designed to identify direct targets, indirect interaction sites, and associated regulatory networks. This service not only reveals the mechanism of action of molecules but also provides functional validation and biological significance analysis, making it an essential component of modern drug discovery and life sciences research.


Target identification is a crucial step in drug development, enabling researchers to determine the targets of small molecules or drugs, reduce off-target effects, and optimize drug design. It addresses several core challenges: first, target identification reveals the interactions between small molecules and disease-associated proteins, providing a molecular foundation for disease mechanism research. Second, it deciphers the roles of post-translational modifications (PTMs) in functional regulation, supporting the advancement of precision medicine. Third, by comprehensively identifying novel or unknown targets, it accelerates biomarker discovery and the development of innovative drugs. Target identification service not only drives basic research but also provides scientific evidence for drug screening, optimization, and preclinical studies.

 

1. Target Identification by Labeled Method

Labeled methods are the primary approaches for target identification. Chemical probes typically consist of a tag (e.g., biotin, bioorthogonal reaction groups, photo-labeling groups, or degrader-related groups), a linker, and an active drug fragment. Compound-centric chemoproteomics (CCCP) and activity-based protein profiling (ABPP) are the most commonly used methods for studying small molecule drug targets. Their mechanism relies on the active group fragment of the small molecule binding tightly to its intended target, while the reporter group fragment effectively labels the biological target. Subsequently, affinity purification techniques are used to enrich proteins selectively bound by the probe. Following this, the targets can be identified using various techniques, including gel electrophoresis and mass spectrometry-based proteomics.

 



Li, G. et al. Front Chem. 2021.

Figure 1. The Composition of a Chemical Probe

 

1.1 Target Identification by CCCP

CCCP originates from classical drug affinity chromatography, a technique that has been used for decades. With the advancement of proteomics technologies, CCCP combines traditional methods with modern proteomics to identify protein targets of small bioactive molecules at the proteome level. Unlike ABPP, the first step in CCCP involves immobilizing the drug molecule onto a matrix, such as magnetic beads or agarose beads. Similar to ABPP, immobilization should not affect the pharmacological activity of the target drug. Subsequently, cell or tissue lysates are incubated with the affinity matrix, followed by extensive washing to remove non-specific binders. After thorough elution, the enriched proteins are identified using proteomics methods. It is also essential to validate target information and the corresponding pharmacological effects. As mentioned above, chemoproteomics approaches have many advantages, such as being unbiased and enabling high-throughput analysis at the proteome level. However, they also have limitations. Using chemoproteomics, in addition to true target proteins, non-specific binding proteins and active metabolites may also be identified, potentially leading to false-positive results. Moreover, proteins that are insoluble in the buffers used during target enrichment (e.g., PBS, Tris-HCl) may pass through the matrix unnoticed. When comparing the two chemoproteomics methods, CCCP cannot detect the activation state of identified proteins as ABPP can. However, CCCP is a more unbiased method that can even identify targets without enzymatic functions, facilitating the discovery of novel targets.

 



Zou, M. et al. Biology (Basel). 2024.

Figure 2. Workflow of Target Identification by CCCP

 

1.2 Target Identification Based on ABPP

ABPP is a technique that combines active probes with proteomics technologies to identify protein targets of small molecules with biological activity, helping to elucidate their mechanisms of action and potential side effects. In a typical ABPP experiment, probes derived from the parent molecule are designed and synthesized based on structure-activity relationship (SAR) studies of the parent compound. The probes should be synthesized as follows: (i) the probe should retain the pharmacological activity of the parent molecule to ensure the accuracy of subsequent target identification; (ii) the probe should facilitate the enrichment of the bound protein targets. Next, the probe is incubated with live cells, lysates, or tissue homogenates to allow full binding to the target proteins. After chemical and biochemical enrichment, the protein targets are identified using proteomics methods. The final step involves validating the target information through techniques such as SPR, MST, or ITC, and confirming the corresponding pharmacological effects through appropriate biological functional analyses.

 



Zou, M. et al. Biology (Basel). 2024.

Figure 3. Workflow of Target Identification by ABBP

 

2. Target Identification by Label-Free Method

Label-free methods utilize small molecules in their native state without any chemical modifications, preserving their natural conformation and functional properties. This approach is often favored by researchers as it eliminates the need to modify or label the primary molecule. While this method avoids any potential issues associated with compound labeling, it has certain limitations. Label-free molecules can bind to non-target proteins, potentially leading to the identification of false-positive targets.

 

2.1 Target Identification by DARTS

Drug affinity responsive target stability (DARTS) is a technique developed based on the principle that small molecules can bind to and stabilize their target proteins, thereby increasing their resistance to proteolysis (i.e., degradation by proteases). DARTS utilizes this property to identify target proteins by detecting an increase in protease resistance induced by binding. In this method, small molecules are incubated with cell lysates and then treated with a protease. If the small molecule binds to its target protein, the protease will be unable to degrade it due to increased stability, resulting in a greater residual protein mass after treatment. The increase in protein stability can be detected using techniques such as Western blotting or mass spectrometry.  DARTS has been employed to identify several protein targets of various small molecules. This technique has proven to be a powerful tool for discovering new small-molecule drugs and understanding the mechanisms of action of these compounds.

 

2.2 Target Identification by SPROX

Denatured proteins are more susceptible to oxidation compared to native proteins. The stability of proteins from rates of oxidation (SPROX) method leverages this characteristic to measure the oxidation rate of surface-exposed methionine residues in proteins, both in the presence and absence of small molecules, and to detect any changes caused by small molecule binding that stabilizes target proteins. In this method, small molecules are incubated with cell lysates, followed by chemical denaturation and the addition of an oxidizing agent (H₂O₂). The protein oxidation rates are then quantified using mass spectrometry. SPROX is applicable only to proteins containing methionine residues, as it identifies target proteins by measuring the oxidation levels of methionine residues. Therefore, SPROX may not be suitable for identifying target proteins that lack methionine residues. It is worth noting that both SPROX and DARTS methods are applicable to cell lysates rather than live biological systems. As a result, they can only study proteins isolated from cells, not intracellular proteins, which may limit their applicability to certain research questions.

 

2.3 Target Identification by CETSA

Cellular thermal shift assay (CETSA) was developed based on the concept of ligand-induced thermodynamic stabilization of protein targets. The thermal stability of a protein can be assessed to evaluate the increase in its stability after ligand binding. CETSA can be performed in live cells or cell lysates. To conduct CETSA, cells or cell lysates are treated with a small molecule or a vehicle control and then heated. Western blotting is used to determine whether proteins denature in a temperature-dependent manner and whether the melting curves of certain proteins change upon interaction with small molecules in the sample. By comparing the thermal stability of proteins with and without the small molecule, it is possible to determine if the small molecule interacts with the protein and to estimate the binding affinity. Although this method relies on Western blotting and may be limited by antibody availability, several high-throughput thermal shift approaches have been developed to identify protein targets, such as MS-CETSA, HCIF-CETSA, and ITDR-MS-CETSA.

 



Tabana, Y. et al. BMC Biotechnol. 2023.

Figure 4. Workflow of Target Identification by Label-Free Method (A-by DARTS, B-by SPROX and C-by CETSA)

 

Service at MtoZ Biolabs

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider, provides advanced proteomics, metabolomics, and biopharmaceutical analysis services to researchers in biochemistry, biotechnology, and biopharmaceutical fields. Our ultimate aim is to provide more rapid, high-throughput, and cost-effective analysis, with exceptional data quality and minimal sample consumption. leveraging advanced chemoproteomics technologies and high-resolution mass spectrometry platforms (such as Orbitrap and Q Exactive HF) , MtoZ Biolabs provides comprehensive target identification service to help researchers accurately analyze the interactions between drugs and targets. Our target identification service includes both labeled and label-free methods. Combined with bioinformatics analysis and functional validation experiments, we comprehensively identify drug targets, analyze post-translational modifications (PTMs), and uncover protein functions and related pathways.  

 

Service Advantages

MtoZ Biolabs' target identification service offers the following advantages:

1. Advanced Chemical Proteomics Technology: MtoZ Biolabs employs labeled and label-free approaches to accurately identify potential targets of natural medicines. With our efficient technology platform, we can deeply explore drug-protein interactions and provide comprehensive target data.

2. High Sensitivity and Specificity: Leveraging mass spectrometry and high-throughput screening technologies, MtoZ Biolabs' target identification service can accurately capture and identify low-abundance proteins in complex biological samples. Our service ensures high sensitivity and specificity, enabling the effective identification of disease-related targets and advancing drug development and precision medicine.

3. Customized Service and Full Support: MtoZ Biolabs offers flexible and customized target identification services covering the entire process from target screening to final validation. Whether for natural or small-molecule drugs, we optimize the service based on specific needs, ensuring precise and valuable data support at every stage of the research.

 

Case Study

1. Development of Photolenalidomide for Cellular Target Identification

Lenalidomide (Len), a thalidomide analog, is a clinically used therapeutic agent that can alter substrate binding of cereblon (CRBN), the substrate receptor of the CRL4 E3 ubiquitin ligase. Here, researchers report the development of photo-lenalidomide (pLen), a Len probe with photoaffinity labeling and enrichment handles, designed for target identification through chemoproteomics. pLen retains the substrate degradation profile, phenotypic antiproliferative, and immunomodulatory properties of Len, and enhances interaction with the CRBN thalidomide-binding domain, as revealed by binding site localization and molecular modeling. Using pLen, researchers captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by pLen and forms a ternary complex with CRBN in the presence of Len in several epithelial cell lines, but is not ubiquitinated or degraded itself. These data suggest that a broader set of CRBN ligand-induced targets may or may not be degraded, which can be identified by applying pLen highly translatably to other biological systems.

 



Lin Z, et al. J Am Chem Soc. 2022.

Figure 5. Development of Photolenalidomide for Cellular Target Identification

 

2. Highly Effective Identification of Drug Targets at the Proteome Level by pH-dependent Protein Precipitation

A thorough understanding of a drug's target space is crucial for studying its mechanisms of action and side effects, as well as for drug discovery and repurposing. In this study, researchers proposed an energetics-based method called pHDPP for probing ligand-induced protein stability shifts to enable proteome-wide drug target identification. They demonstrated that pHDPP is applicable to various ligands, including folate derivatives, ATP analogs, CDK inhibitors, and immunosuppressants, thereby enabling highly specific target identification of cell lysates. This method was compared with thermal and solvent-induced denaturation methods using pan-kinase inhibitors as model drugs, demonstrating its high sensitivity and strong complementarity with other methods. Dihydroartemisinin (DHA) is the primary artemisinin derivative used to treat malaria and is known to have remarkable effects in the treatment of various cancers. However, its antitumor mechanism remains unknown. Applying pHDPP to reveal the target space of DHA, 45 potential target proteins were identified. Pathway analysis indicated that these target proteins are mainly involved in metabolic and apoptotic pathways. Two cancer-related target proteins, ALDH7A1 and HMGB1, were validated through structural modeling and AI-based target prediction methods. They were further confirmed to have strong affinity for DHA through CETSA. In summary, pHDPP is a powerful tool for constructing the target protein space to reveal drug mechanisms of action, with wide applications in drug discovery research.

 



Zhang, X. et al. Chem Sci. 2022.

Figure 6.  Workflow of the pH Dependent Protein Precipitation (pHDPP) Approach and the Investigation of the Acidic Agents for Drug Target Identification

 

Applications

Target identification service can be used in:

1. Drug Target Discovery: Target identification elucidates the mechanism of action of drugs and providing a basis for drug development.  

 

2. Drug Development and Optimization: Target identification optimizes drug efficacy and selectivity based on target information.  

 

3. Disease Mechanism Analysis: Revealing key pathways associated with diseases through target research.  

 

4. Discovery of Novel Biomarkers: Target identification discovers potential diagnostic and therapeutic targets by leveraging the functional properties of drugs.

 

Deliverables

1. Comprehensive Experimental Details

2. Materials, Instruments, and Methods

3. Relevant Liquid Chromatography and Mass Spectrometry Parameters

4. The Detailed Information of Target Identification

5. Mass Spectrometry Image

6. Raw Data

 

MtoZ Biolabs' target identification service provides robust technical support for drug development, disease mechanism research, and biomarker discovery, accelerating research progress and driving breakthroughs in life sciences. If you are interested in our service, please feel free to contact us.