Table 11.

Benefits and drawbacks of each target identification approach.

Approach	Benefits	Drawbacks
Affinity chromatography	1. The synthesis of immobilized probes is simple. 2. It facilitates the analysis of all adsorbed proteins without discrimination. 3. It can be easily conducted in individual laboratories.	1. Discrimination between truly high-affinity target proteins and high-abundance proteins with low affinity is necessary. 2. The elution process relies on empirical knowledge; excessively strong washing conditions can significantly reduce the number of identified targets, while weak elution conditions can yield false-positive results. 3. It does not reveal interactions between drugs and proteins in live cells under physiological conditions. 4. Biotin affinity tags introduce steric hindrance, potentially reducing or even abolishing the activity of natural active compounds and impeding probe molecule entry into cells.
ABPP	1. It exhibits strong specificity. 2. ABPs do not require pre-immobilization on matrix material. 3. It can be directly applied to live cells, elucidating drug–target interactions under physiological conditions.	1. Only proteins capable of forming covalent interactions with bioactive natural products can be captured. 2. Synthesizing ABPs relies on the structure of the bioactive natural product and necessitates expertise in medicinal chemistry.
PAL	1. It can facilitate the analysis of proteins that cannot be targeted by ABPs.	1. Photoaffinity groups still exhibit non-specific binding to abundant cellular proteins. 2. The synthesis of PAL probes necessitates expertise in medicinal chemistry.
Proteome microarray assay	1. It requires minimal sample volumes. 2. High throughput enables parallel analysis of thousands to millions of proteins. 3. Utilizing protein chips is relatively simple.	1. It is relatively costly, necessitating a range of expensive, high-end instruments. 2. Standardization issues with the chips.
DARTS	1. No structural modification of natural bioactive compounds is necessary. 2. It can be easily implemented by individual laboratories.	1. Certain natural products do not significantly affect the conformation of the target protein upon binding. 2. Some proteins are less susceptible to proteases, resulting in undetectable changes in DARTS. 3. The ability to identify targets that exhibit low protein abundance is limited.
TPP	1. It monitors changes in protein thermal stability throughout the proteome during the action of natural bioactive compounds. 2. It enables high-throughput detection of target proteins.	1. Inapplicable to heat-insensitive proteins. 2. The extraction of thermodynamic parameters besides the Tm value from TPP data is challenging. 3. The magnitude of the Tm shift after protein binding with natural active compounds does not consistently correlate with binding affinity. 4. Time-consuming and costly.
SPROX	1. It enables the large-scale assessment of protein folding states and potentially precise determination of binding domains and peptide segments involved in the interactions between compounds and target proteins.	1. It can only detect binding between proteins containing methionine residues and small molecules. 2. Generating the standard curve requires a large sample volume. 3. The process is complex, costly, and requires substantial resources.
CETSA	1. No structural modification of natural bioactive compounds is required. 2. It can be easily implemented by individual laboratories.	1. Detection necessitates relatively high concentrations of the natural active compound. 2. The process exhibits low throughput in discovering new targets and is primarily utilized for target validation.
ITC	1. It provides fast, accurate, and low-sample-volume analysis with minimal requirements for reaction system transparency, turbidity, and viscosity. 2. It enables precise determination of comprehensive thermodynamic information.	1. It is typically utilized as an auxiliary screening technique in high-throughput screening due to its low-throughput nature. 2. It requires specialized ITC equipment.