Probing small molecule–protein interactions: A new perspective for functional proteomics

Abstract

The isolation of proteome subsets on the basis of the interactions of small molecules with proteins is an emerging paradigm in proteomics. Depending on the nature of the small molecule used as a bait, entire protein families can be monitored in biological samples, or new functions can be attributed to previously uncharacterized proteins. With pharmaceutical compounds as baits, drug targets and toxicity-relevant off-targets can be discovered in an unbiased proteomic screen. At the heart of this strategy are synthetic bi- or trifunctional small molecule probes. These probes carry the small molecules of interest as baits (selectivity function), as well as a sorting function for the isolation of small molecule–protein complexes or conjugates from complex protein mixtures. In some designs, a covalent linkage of the bound protein to the probe is established through a separate reactivity function or a combined selectivity/reactivity function. The covalent linkage allows for isolation or detection of probe–protein conjugates also under harsh or denaturing conditions. Ultimately, specifically isolated proteins are commonly identified by mass spectrometry. This review summarizes probe designs, workflows, and published applications of the three dominant approaches in the field, namely affinity pulldown, activity-based protein profiling, and Capture Compound Mass Spectrometry.

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.