Click Chemistry in Proteomic Investigations

Abstract

Despite advances in genetic and proteomic techniques, a complete portrait of the proteome and its complement of dynamic interactions and modifications remains a lofty, and as of yet, unrealized, objective. Specifically, traditional biological and analytical approaches have not been able to address key questions relating to the interactions of proteins with small molecules, including drugs, drug candidates, metabolites, or protein post-translational modifications (PTMs). Fortunately, chemists have bridged this experimental gap through the creation of bioorthogonal reactions. These reactions allow for the incorporation of chemical groups with highly selective reactivity into small molecules or protein modifications without perturbing their biological function, enabling the selective installation of an analysis tag for downstream investigations. The introduction of chemical strategies to parse and enrich subsets of the "functional" proteome has empowered mass spectrometry (MS)-based methods to delve more deeply and precisely into the biochemical state of cells and its perturbations by small molecules. In this Primer, we discuss how one of the most versatile bioorthogonal reactions, "click chemistry", has been exploited to overcome limitations of biological approaches to enable the selective marking and functional investigation of critical protein-small-molecule interactions and PTMs in native biological environments.

Figure 1.. Biological studies enabled by proteomics.

Highlighted in orange are applications that can be integrated with chemical methods to aide in proteomic investigations

Figure 2.. General strategies to introduce bioorthogonal functionality to study proteins.

Step 1) Bioorthogonal reactive groups are first introduced onto protein targets through one of two general strategies, either i) direct chemical modification of endogenous proteins with chemical probes (top path), or ii) through a genetic tag, such as unnatural amino acid mutagenesis with functionalized amino acids and orthogonal tRNA/AARS pairs or enzymatic ligation of acceptor peptides with bioorthogonal motifs on engineered enzymes (bottom path). Step 2) Chemical tags possessing complementary bioorthogonal groups are selectively ligated to modified proteins. Step 3) Marked proteins are subsequently visualized, quantified and/or identified using a variety of techniques.

Figure 3.

Common bioorthogonal chemical reactions. Red and blue circles correspond to the two “reactants” of a bioorthogonal reaction: 1) proteins labeled with a bioorthogonal reactive group and 2) complementary reactive tag for protein identification and/or visualization. Highlighted in yellow are the bioorthogonal reactive groups (left) and corresponding ligation product (right).

Figure 4.

General “bottom-up” proteomics experimental workflow.

Figure 5.

Chemoproteomic methods to map features of proteins or to characterize molecular interactions of bioactive compounds using chemical probes. (A) General design of click probes used in protein-centric or chemical-centric proteomic investigations. (B) Schematic of proteomic workflows. (C) Various readouts of chemoproteomic investigations.

Figure 6.

Examples of click probes chemical proteomic investigations

Figure 7.

General classes of click-chemistry methods for the identification of post-translational modifications. a) The chemical reactivity of certain PTMs allows them to be reacted directly and selectively with bioorthogonal probes. b) The metabolism of living cells or animals can be exploited for the introduction of chemical probes with click-chemistry handles. c) Other PTMs can be modified enzymatically to install click-chemistry handles.

Figure 8.

Chemical mechanisms responsible for the selective reaction of sulfenylated and sulfinylated cysteine residues with their respective probes.