The 2022 Nobel Prize in Chemistry for the development of click chemistry and bioorthogonal chemistry

Abstract

The 2022 Nobel Prize in Chemistry recognized the development of biorthogonal chemical ligation reactions known as click chemistry in biomedicine. This concept has catalyzed significant progress in sensing and diagnosis, chemical biology, materials chemistry, and drug discovery and delivery. In proteomics, the ability to incorporate a click tag into proteins has propelled development of powerful new methods for selective enrichment of protein complexes that inform understanding of protein networks. It also has had a strong influence on the ability to enrich for protein post-translational modifications. This feature article summarizes the impacts of biorthogonal click chemistry on proteomics.

Introduction

In 2002, the research groups of Kolb et al. [1] and Tornøe et al. [2] independently published the copper-catalyzed azide-alkyne cycloaddition as a chemical ligation reaction (Fig. 1A) now known and in widespread use as click chemistry. This concept whereby molecular building blocks become ligated quickly and efficiently has had widespread influence on fields including chemical biology [3], drug delivery, gene expression, real-time imaging, and proteomics [4, 5]. The investigators were awarded the 2022 Nobel Prize in Chemistry in recognition of the importance of biorthogonal ligation reactions. This feature article summarizes the applications of click chemistry in proteomics.

Fig. 1.

Click chemistry used in proteomics

Bioorthogonal reactions selectively modify proteins in native biological environments using functional groups absent in living systems that do not react with endogenous biomolecules and undergo specific, controllable, high-yield ligation reactions (Fig. 2). Mahal et al. [6] demonstrated that biosynthetic installation of a unique chemical entity, in this case a ketone, on the cell surface serves as a molecular handle for specific addition of a fluorescent tag (Fig. 1B). For this example, they took advantage of the permissiveness of the sialic acid biosynthetic machinery to accept unnatural mannosamine precursors in which the N-acetyl group was substituted with a ketone-containing levulinoyl group. They then attached a biotin group via hydrazide chemistry and used an avidin-conjugated fluorescent tag to visualize the cells using flow cytometry. They applied this reaction to alter the glycosylation patterns on cells, target tumors, and provide receptors for viral-mediated gene transfers. However, the usefulness of ketone substitution was limited by the presence of endogenous keto-metabolites that competed with the bioorthogonal ligation reaction.

Fig. 2.

General strategies to functionalize cellular proteins using click chemistry

Saxon and Bertozzi [7] then realized that use of azido sugar intermediates would allow the biosynthetic machinery to add azido groups to cell surface sialic acids. Such modified sugars could be ligated under biological conditions using a biotinylated phosphine designed for water solubility. This solved the problem of endogenous nonspecific amine acylation and could be performed in living organisms to remodel cell surface glycosylation [8]. Because the phosphines did not deliver ideal aqueous reactivity, the investigators sought to exploit the azide-alkyne cycloaddition reaction (Fig. 1A). However, the copper catalyst is toxic, precluding its use in applications where cells must remain viable. Agard et al. [9] used a strained cyclooctane ring to activate the alkyne without the necessity of a copper catalyst without apparent cellular toxicity (Fig. 1C). They demonstrated this approach for in vivo imaging of membrane-associated glycans in developing zebrafish [10]. These investigators also developed a metabolic oligosaccharide engineering approach whereby the use of membrane-permeable monosaccharide precursors with chemical tags enables biorthogonal chemistry [11].

Use of click chemistry in proteomics applications

Traditional proteomics infers the abundances of gene products from those of tryptic peptides measured in mass spectrometry experiments. Because of the presence of splice variants, amino acid substitutions, post-translational modifications, and chemical modifications that are often not distinguished by proteolytic peptides, bottom-up proteomics identifies protein groups consisting of proteoforms [12]. With the use of multidimensional chromatography and state-of-the-art liquid chromatography–mass spectrometry, one can quantify the complete proteome, defined as approximately 10,000 protein groups for a given cell type [13]. To add functional information on the biochemical pathways enriched in each biological system, the protein groups observed to undergo abundance changes are grouped using gene set enrichment analysis according to curated gene lists, giving the user an idea of pathway-level changes that occur during the biological process [14]. To infer further detail, investigators look at changes in post-translational modifications, particularly for the flux in protein phosphorylation associated with signal transduction and other cellular processes [15]. Analysis of protein complexes provides data that inform network modeling that have the potential to identify actionable drug development targets. One widely cited approach is to use genetic constructs for bait proteins of interest with genetically encoded affinity handles [16]. To address the challenge of differentiating specific from nonspecific binding events, genetic constructs with tandem affinity tags are often used [17].

Proximity labeling entails the use of a genetic construct that codes for an enzyme that localizes to a cellular compartment of interest and executes a biological transformation (such as biotinylation) that can be used as an affinity handle for the enrichment of functionally associated proteins (Fig. 2) [18]. This approach enriches the proteins modified with the affinity handle within a known molecular radius, circumventing concerns about the extent to which weak binding interactions survive traditional affinity purification.

As reviewed by Parker and Pratt [11], bioorthogonal reactions allow proteomics investigators to incorporate chemical groups into biological molecules with high reaction selectivity. They use these reactions for (i) selective capture of interacting proteins and small molecules under native conditions; (ii) capture for direct assessment of protein network interactions; and (iii) enrichment of proteins with specific modifications [11].

Selective capture of interacting proteins and small molecules under native conditions

Many alkylating agents and chemoproteomics probes have been modified to include a click handle to facilitate enrichment in proteomics experiments. In shotgun proteomics, investigators often alkylate cysteine residues to prevent further reactivity. But the incorporation of a click tag in the iodoacetamide structure enables subsequent attachment of a biotin handle for enrichment and prediction of functional cysteines in proteomes [19]. Click tags can also be incorporated through unnatural amino acid mutagenesis that incorporates functionalized amino acids. It is also possible to incorporate bioorthongal tags using enzymatic ligation reactions (Fig. 2).

Investigators have designed chemical probes that contain a recognition group for target protein(s) of interest, an electrophilic or photoreactive group that binds interacting proteins, and a reporter tag containing an affinity handle, fluorophore, or click tag (Fig. 2). These click-enabled probes allow efficient enrichment of low-abundance interacting proteins and/or trapping of probe-protein interactions in living cells. Click chemistry potentiates chemoproteomics probes that target enzyme classes by eliminating the need for bulky reporter tags that may interfere with cellular processes. Such sterically minimized click tags overcome these challenges and provide convenient enrichment or detection systems compatible with living cells. The use of click functionality has also been employed to enhance the membrane permeability of probes for chemoproteomics studies [20]. Click probes have been employed to target other nucleophilic amino acid reactivity, including serine, lysine, tyrosine, and methionine [11].

Capture for direct assessment of protein network interactions

Investigators have employed genetically encoded handles containing one or two affinity groups with the aim to characterize interaction networks of target proteins at the proteome scale [17]. While affinity-based purification works well for high-affinity interactions, it is not effective for the study of low-affinity interactions. Photoaffinity labeling was developed to solve this problem by fixing transient reversible interactions upon light irradiation. As reviewed by Lapinsky and Johnson [21], clickable photoprobes provide an efficient means of purifying photo-linked protein complexes. But because most proteins still lack specific chemical tools to study their functions, chemical proteomics investigators are characterizing libraries of functionalized chemical probes that possess click functionality for target capture and enrichment.

Enrichment of proteins with specific post-translational modifications

Post-translational modifications diversify the structures and functions of the proteome and are therefore important targets for proteomics studies [22]. Enzymes that add PTMs may be considered as writers and those that remove them as erasers. Proteins that contain modules that bind to a PTM may be regarded as reader enzymes. The complexity of these interactions increases with the complexity of eukaryotic life forms [23]. Because PTMs are often sub-stoichiometric, it is often necessary to use targeted enrichment to facilitate their profiling using proteomics (Fig. 3A).

Fig. 3.

Click chemistry workflows used for PTM-proteomics

Glycoproteomics

The O-GlcNAc modification present on intracellular proteins occurs to many of the same Ser and Thr residues that are phosphorylated [24]. The discovery of this modification was delayed by decades since it cannot be specifically radiolabeled. In addition, O-GlcNAc is chemically labile due to a facile beta-elimination reaction that makes mass spectrometric identification of sites of modification challenging. To facilitate enrichment, investigators have employed and engineered galactosyltransferase that attaches an azido-tagged GalNAc residue to O-GlcNAc-modified peptides. Subsequent reaction with a photo-cleavable biotin enrichment tag enables direct proteomics identification of modified peptides (shown conceptually in Fig. 3B) [25].

Complex protein glycosylation occurs via a conserved biosynthetic pathway in the endoplasmic reticulum and Golgi apparatus [24]. Because the biosynthetic reactions do not go to completion, the resulting glycosylation at each protein site is heterogeneous, diversifying the lectin-containing binding partners with which they interact. To predict the biological functions of glycoproteins, it is therefore important to quantify the population of glycoforms that exist at each glycosite. This poses a challenge to traditional bottom-up glycoproteomics because glycan masses and heterogeneity cannot be predicted directly from the genome sequences that inform proteomics database searches. Also, complex glycosylation alters the dissociation patterns produced by collisional activation tandem mass spectrometry, resulting in the need for specialized glycoproteomics search engines [26].

Woo et al. [27] developed an isotope-targeted glycoproteomics reagent (IsoTaG) (Fig. 1D) that contains a pair of bromine atoms that confer a unique isotopic signature following biorthogonal tagging. The tag consists of an alkyne group for click ligation, a dibrominated isotope site, a photocleavage site, a linker, and a biotin group. Thus, the investigators enriched the tagged glycopeptides, cleaved the linker, and then used the isotopic signature to identify glycopeptides in the tryptic digest.

Lipidomics

Because antibody enrichment tools are not available, investigators have incorporated lipids containing biorthogonal functionalities using the cellular metabolic enzymes [28] that have enabled the discovery of key lipidation events that correlate with disease states. Anti-acetyl lysine antibodies have served as powerful tools for the enrichment of protein acetylation sites in proteomics studies. Because such antibody tools are not well suited for the analysis of acetylation dynamics, bioorthogonal reporter probes have been developed to distinguish between enzymatic acetylation reactions and those created by acetyl-CoA-mediated chemical alkylation [29]. While antibodies that recognize methylated lysines and arginines are available, bioorthogonal chemistry has enhanced the ability to study sub-classes of protein methyl transferase enzymes [30]. Investigators used a similar strategy to create probes for specific family members of the poly-ADP-ribose polymerase enzymes that ADP-ribosylate protein lysine, asparagine, glutamine, serine, and cysteine (31).

Conclusions

The 2022 Nobel Prize in chemistry recognized the sustained impact that biorthogonal click chemistry has had on numerous biomolecular fields. In proteomics, the power and convenience of click chemistry to target protein enrichment is now a widely used tool that potentiates the uses of genomic information for understanding the roles of protein proteoforms in biological mechanisms.

Biography

Joseph Zaia has published more than 150 articles in peer-reviewed scientific journals and has an H-index of 55. He is an editor of Analytical and Bioanalytical Chemistry. His research group focusses on the analytical biochemistry of glycoproteins and proteoglycans. They have developed methods for combined glycomics, proteomics, and glycoproteomics from tissue slides. They have produced a set of bioinformatics tools for the interpretation of mass spectrometry–based glycomics and glycoproteomics data sets. Their major goals are to track changes in protein glycosylation during biological mechanisms. Specific projects include (i) mapping changes in extracellular matrix protein glycosylation during brain disease mechanisms and (ii) analysis of the roles of viral spike protein glycosylation in the evolution of pathogenic enveloped viruses.