Probing small molecule–protein interactions: A new perspective for functional proteomics
Graphical abstract
Highlights
► Modular chemical probes enable the isolation of subproteomes defined by functional small molecule-protein interactions. ► Leading strategies are Activity-based protein profiling, affinity pulldown, and Capture Compound mass spectrometry. ► Profiling small molecule-protein interactions contributes to the functional annotation of genomes. ► The approaches enable unbiased direct proteomic screens to identify all proteins that interact with a given drug molecule.
Introduction
Diversification of the experimental approaches with a shift from complex unfractionated protein samples to the investigation of subproteomes has been a general trend in proteomics over the past decade [1]. This allowed identification of lower abundant proteins, and the assignment of some of the functional properties of the proteins, depending on the sample preparation workflow. Examples are the focused analysis of protein sets that share certain types of posttranslational modifications, such as phosphoproteins and their phosphorylation sites in an approach termed “phosphoproteomics” [2]. Other examples are the assignment of proteins to particular subcellular structures, such as nuclei, mitochondria, etc., in an approach termed subcellular proteomics [3], or the large-scale assessment of protein–protein interactions [4]. The common rationale behind these types of analyses was to develop the concept of proteome mapping further towards functional proteomics (see, e.g., [5]). The interactions of metabolites, co-factors, or drug molecules with proteins particularly reveal protein function. It is, therefore, an emerging approach to isolate functional subproteomes on the basis of the interactions of proteins with small molecules (Fig. 1).
Functional subproteome isolation by means of small molecules is associated to the field of chemical proteomics. Chemical proteomics encompasses the profiling of small molecule–protein interactions using small molecule probes (which is the focus of this review article), but also includes molecular biology-driven strategies or small molecule microarrays. Exemplary recent reviews of this wider range of technical approaches, have been provided by Ovaa [6] and Uttamchandani and Yao [7], [8]. Also, chemical proteomics covers the use of synthetic cofactor or substrate analogs to track posttranslational modifications. In this approach, sometimes termed ‘catalomics’ [9], the probes are substrate analogs of the modifying enzymes, and the aim is to identify the acceptor proteins for the respective modification. Published studies addressed methylation [10], [11], palmitoylation [12], acetylation [13], glycosylation [14], phosphorylation [15], [16], or farnesylation [17], [18] (see [9], [19], [20], [21], [22] for recent reviews). Protein targets of disease-relevant modifications such as α, β unsaturated aldehydes generated during free radical-induced damage of polyunsaturated fatty acids have been studied in this way as well [23]. However, a more thorough discussion of this approach is beyond the scope of this review.
Section snippets
Small molecule probes: rationale and design modules
The rationales for designs and functionalities of probes for small molecule–protein interaction profiling comprise elements of affinity chromatography (a key technique in protein purification [24]), photo-affinity labeling (to detect and characterize small molecule binding to target proteins [25]), and active-site chemical labeling of enzymes (see, e.g., [26]). These techniques are traditionally aimed to specifically target and characterize individual proteins. The scope of the newly devised
Small molecule–protein interaction profiling for the functional annotation of the genome and comparative proteomics
In this section, studies are reviewed that aim at the profiling of known and previously unknown members of protein families that share the binding to a particular small molecule. The small molecule of interest is used as the selectivity function of affinity pulldown probes, ABPP or Capture Compounds. ABPP selectivity functions, as outlined above, act as suicide inhibitors through a chemical reaction within enzyme active sites. Thus, the probe is specifically attached via the enzymatic catalysis
Small molecule–protein interaction profiling in drug discovery
One of the most significant foreseeable roles for chemical proteomics and in particular for profiling small molecule–protein interactions is in the field of drug discovery [29], [64], [117]. Through the profiling of interactions between small molecule drugs and proteins, several key questions in drug discovery have been shown to be addressable: (a) Identification of the proteins that underlie the drug mode of action. Drug target proteins can be directly identified and their binding affinities
Conclusion
Already at the present stage of experimental studies that have been conducted, small molecule–protein interaction profiling has emerged as a powerful strategy in proteomics. Functions can be assigned to previously non-annotated gene products, and protein classes assessed in great depth in biological samples. The different technical approaches regarding the design of the small molecule probes each have specific strengths and limitations. While in ABPP, with mechanism-based labeling probes, the
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T.L. and J.J.F contributed equally to this work.