Proteins are the fundamental components of the cell and their three-dimensional structures have continuingly evolved to fit their specific functions in different cellular processes. The biochemical functions of a protein always involve interactions with diverse ligands, such as substrates, inhibitors, signaling molecules or allosteric modulators, etc. [1, 2, 3, 4, 5, 6] In response to the ligand binding, protein conformation is affected, inducing an alteration in enzyme activity or transducing signals to downstream effectors. Identifying protein–ligand interactions is important to reveal the regulatory mechanisms of proteins as well as the action mechanisms of ligands [4, 5, 6]. In addition to the determination of the ligand-binding proteins, identification of the ligand-binding sites is also extremely necessary [7]. Therefore, it is of great interest to develop robust approaches to uncover the binding relationship between ligands and their targeting proteins, as well as their exact binding sites.

Affinity probe–based method is a classical chemical proteomics approach, in which the immobilized or tagged small molecules are incubated with cell lysates for capturing the interacting proteins [8,9]. It has been successfully applied to identify the target kinases by using the immobilized nonselective kinase inhibitors (kinobeads) [10,11]. However, the affinity probe–based method requires chemical modification of the small molecules, which might alter the binding specificity of the ligands. Therefore, alternative screening approaches are also needed. The modification-free approaches have the advantages of bypassing chemical modification of ligands and thus can be used to identify the target proteins of diverse ligands at the proteome level. There are already some other reviews [7,12] that describe modification-free approaches in detail. Herein, in this review, we will briefly introduce the modification-free approaches, focusing on their recent methodology advancements in identifying ligand–protein interactions.