Introduction
In recent years, substantial advancements in genetic and proteomic techniques have catapulted our understanding of biological systems, particularly the proteome. However, despite these advancements, capturing the dynamic nature of protein interactions and post-translational modifications (PTMs) remains a significant challenge. Traditional biological and analytical approaches fall short in key areas, notably the interactions of proteins with small molecules and various PTMs. Fortunately, the emergence of bioorthogonal chemistry, especially "click chemistry", has paved the way to overcome these hurdles and provide deeper insights into the functional state of proteins within their native environments.
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Fig. 1 General classes of click-chemistry methods for the identification of post-translational modifications (PTMs) (Parker C. G., Pratt M. R. 2020).
Advances in Proteomic Techniques
Explosive advances in genome sequencing technologies have significantly augmented our understanding of the proteome, offering insights into physiological processes and disease mechanisms. However, proteomes are unique in their temporal and contextual variability, which sets them apart from the relatively stable genomes. This variability poses a challenge for genome-based strategies to study proteins and their interactions. Proteomic methods, particularly those utilizing MS, are pivotal as they enable the measurement of various protein properties, including abundance, tissue distribution, subcellular localization, and PTMs, along with protein-protein interactions (PPIs).
A significant portion of MS-based proteomics employs a bottom-up workflow, commonly termed "shotgun proteomics". Here, proteins are identified and quantified by analyzing peptides generated from proteolysis. The process involves extraction and digestion of proteins followed by liquid chromatography to separate peptides, which are subsequently analyzed by MS. Database searching tools like Sequest or Mascot facilitate the identification and quantitation of proteins. This traditional approach, although powerful, often falls short in mapping dynamic protein interactions and modifications.
Bioorthogonal Chemistry: Bridging the Experimental Gap
To overcome the limitations posed by traditional proteomic techniques, chemists have introduced bioorthogonal reactions—chemical reactions that can occur inside living systems without interfering with native biochemical processes. These reactions typically involve functional groups that are not present in living organisms, ensuring they do not react with biological components. One such versatile bioorthogonal reaction is "click chemistry", particularly the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), which enables the selective installation of visualization or enrichment tags onto target proteins.
Click chemistry plays a crucial role in proteomic investigations by enabling the selective labeling and enrichment of proteins in their native environments. For instance, it facilitates the identification and quantitation of small molecule-protein interactions and various PTMs. The process generally involves two steps: first, introducing a bioorthogonal reactive group to the protein of interest, and second, using a complementary reactive group to ligate an analysis tag, such as biotin, that allows for subsequent downstream analyses via MS.
Protein Post-Translational Modifications
PTMs significantly diversify the proteome beyond the static genome by adding functional groups to proteins, influencing their activities and interactions. Proteomic analyses of PTMs require selective enrichment and labeling strategies due to their often substoichiometric and dynamic nature. Click chemistry has proven invaluable in this realm, particularly for studying glycosylation, lipidation, acetylation, methylation, ADP-ribosylation, and cysteine redox modifications.
Glycosylation: One of the Most Common PTMs
Glycosylation, involving the covalent addition of carbohydrates to proteins, is crucial for various biological functions. Traditional methods for glycoprotein analysis relied on lectins and antibodies, which often lack specificity. Click chemistry, particularly metabolic oligosaccharide engineering, has transformed this field. Azide- or alkyne-containing analogs of monosaccharides can be incorporated into glycans by endogenous enzymes, allowing for selective labeling and identification of glycoproteins. Isotope-targeted glycoproteomics (IsoTaG) further enhances the detection of low-abundance glycopeptides, providing insights into glycosylation patterns in different biological contexts.
Lipidation: Controlling Protein Localization
Lipidation regulates protein localization and function across cellular membranes. Traditional visualization methods relied on radioactive lipids, which were cumbersome and limited in scope. Bioorthogonal lipid MCRs have greatly expanded our ability to study lipidation. Metabolic incorporation of azide- or alkyne-modified fatty acids into proteins enables the profiling of lipidated proteins via click chemistry. For instance, alkynyl-lipids have been used to identify myristoylated and palmitoylated proteins, shedding light on their roles in pathogen biology and antiviral responses.
Acetylation: Regulating Gene Transcription and Beyond
Lysine acetylation, mediated by acetyltransferases and deacetylases, plays a key role in gene transcription and other cellular processes. Anti-acetyl lysine antibodies have facilitated the identification of numerous acetylation sites, but they do not capture the dynamic nature of this modification. Click chemistry-based MCRs allow for pulse-chase experiments to study the dynamics of acetylation and to distinguish between enzymatic and chemical modifications of proteins. Additionally, MCRs for other acylations, such as malonylation and succinylation, have been developed, enabling comprehensive profiling of protein acylation.
Methylation: Adding Versatility to Lysine and Arginine Modifications
Methylation adds one to three methyl groups to lysine or arginine residues, influencing various protein functions. Bioorthogonal SAM analogs and engineered MTs have facilitated the identification of methylation sites, though their application is limited to cell lysates. Recent advancements in metabolic engineering have enabled the generation of alkyne-SAM in living cells, broadening the scope of methylation studies.
ADP-Ribosylation: A Dynamic Modification
ADP-ribosylation, mediated by poly-ADP-ribose polymerases (PARPs), is vital for stress response and DNA repair. Bioorthogonal NAD+ analogs and MCRs have been developed to study this complex modification. These tools have revealed specific substrates of PARP family members and enabled the analysis of ADP-ribosylation in living cells.
Cysteine Redox Modifications: A Spectrum of Oxidative Reactions
Cysteine thiols can undergo various oxidative modifications, contributing to the regulation of protein functions. Traditional methods lacked specificity for these modifications, but bioorthogonal probes have provided selective tools for their analysis. For instance, dimedone-based probes have been used to identify sulfenylated cysteine residues, revealing their roles in regulating kinase activity.
Conclusion
The synergy of click chemistry and proteomics has ushered in a new era of biological research. By addressing the limitations of traditional methods, click chemistry has enabled the detailed study of protein interactions and modifications in native environments. This integrated approach holds immense potential for uncovering the complexities of the proteome, paving the way for novel therapeutic strategies and a deeper understanding of cellular biochemistry. As technologies continue to evolve, the combined power of click chemistry and proteomics will undoubtedly yield further transformative discoveries, illuminating the intricate dance of proteins that underpins life itself.
Reference
- Parker C. G., Pratt M. R. Click chemistry in proteomic investigations. Cell. 2020, 180(4): 605-632.
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