Transcriptome-wide identification of RNA-binding proteins (RBPs) is a prerequisite for understanding the posttranscriptional gene regulation networks. However, proteomic profiling of RBPs has been mostly limited to polyadenylated mRNA-binding proteins, leaving RBPs on nonpoly(A) RNAs, including most noncoding RNAs (ncRNAs) and pre-mRNAs, largely undiscovered. Here we present a click chemistry-assisted RNA interactome capture (CARIC) strategy, which enables unbiased identification of RBPs, independent of the polyadenylation state of RNAs. CARIC combines metabolic labeling of RNAs with an alkynyl uridine analog and in vivo RNA-protein photocross-linking, followed by click reaction with azide-biotin, affinity enrichment, and proteomic analysis. Applying CARIC, we identified 597 RBPs in HeLa cells, including 130 previously unknown RBPs. These newly discovered RBPs can likely bind ncRNAs, thus uncovering potential involvement of ncRNAs in processes previously unknown to be ncRNA-related, such as proteasome function and intermediary metabolism. The CARIC strategy should be broadly applicable across various organisms to complete the census of RBPs.
A cell-specific metabolic glycan labeling strategy has been developed using azidosugars encapsulated in ligand-targeted liposomes. The ligands are designed to bind specific cell-surface receptors that are only expressed or up-regulated in target cells, which mediates the intracellular delivery of azidosugars. The delivered azidosugars are metabolically incorporated into cell-surface glycans, which are then imaged via a bioorthogonal reaction.
The copper(I)-catalyzed azide-alkyne cycloaddition, the most widely recognized reaction of click chemistry, is accelerated by tris(triazolylmethyl)amine-based ligands. Here, we compared two new ligands in this family, BTTP and the corresponding sulfated ligand BTTPS, for three bioconjugation applications: (1) labeling of alkyne-tagged glycoproteins in crude cell lysates, (2) labeling of alkyne/azide-tagged glycoproteins on the surface of live mammalian cells, and (3) labeling of azides in surface proteins of live Escherichia coli. Though BTTPS exhibits faster kinetics than BTTP in accelerating the CuAAC in in vitro kinetic measurements, its labeling efficiency is slightly lower than BTTP in conjugating biomolecules bearing a significant amount of negative charges due to electrostatic repulsion. Nevertheless, the negative charge conferred by the sulfate at physiological conditions significantly reduced the cellular internalization of the coordinated Cu(I), thus making BTTPS-Cu(I) a better choice for live cell labeling.
Metabolic labeling of glycans with chemical reproters (e.g., alkyne or azide) in conjunction with bioorthogonal chemistry is a powerful tool for imaging glycome; however, this method lacks protein-specificity and therefore is not applicable to imaging glycosylation of a specific protein of interest (POI). Here we report the development of a cis-membrane FRET-based methodology that allows protein-specific imaging of glycans on live cells. We exploit metabolic glycan labeling in conjunction with site-specific protein labeling to simultaneously install a FRET acceptor and a donor onto the glycans and the extracellular terminal of the protein of interest, respectively. The intramolecular donor-acceptor distance for the POI falls within the range for effective FRET, whereas the intermolecular FRET is disfavored since the excess acceptors on other proteins are distant from the donor. We demonstrated the capability of this cis-membrane FRET imaging method by visualizing the sialylation of several important cell surface receptors including integrin αXβ2, epidermal growth factor receptor, and transforming growth factor-beta receptor type I. Furthermore, our imaging experiments revealed that the sialylation might be important for β2 integrin activation. Our methodology should enable the live-cell studies on how glycosylation regulates the functions and dynamics of various cell-surface proteins.
Large-scale quantification of protein O-linked β- N-acetylglucosamine (O-GlcNAc) modification in a site-specific manner remains a key challenge in studying O-GlcNAc biology. Herein, we developed an isotope-tagged cleavable linker (isoTCL) strategy, which enabled isotopic labeling of O-GlcNAc through bioorthogonal conjugation of affinity tags. We demonstrated the application of the isoTCL in mapping and quantification of O-GlcNAcylation sites in HeLa cells. Furthermore, we investigated the O-GlcNAcylation sensitivity to the sugar donor by quantifying the levels of modification under different concentrations of the O-GlcNAc labeling probe in a site-specific manner. In addition, we applied isoTCL to compare the O-GlcNAcylation stoichiometry levels of more than 100 modification sites between placenta samples from male and female mice and confirmed site-specifically that female placenta has a higher O-GlcNAcylation than its male counterpart. The isoTCL platform provides a powerful tool for quantitative profiling of O-GlcNAc modification.
In the heart, glycosylation is involved in a variety of physiological and pathological processes. Cardiac glycosylation is dynamically regulated, which remains challenging to monitor in vivo. Here we describe a chemical approach for analyzing the dynamic cardiac glycome by metabolically labeling the cardiac glycans with azidosugars in living rats. The azides, serving as a chemical reporter, are chemoselectively conjugated with fluorophores using copper-free click chemistry for glycan imaging; derivatizing azides with affinity tags allows enrichment and proteomic identification of glycosylated cardiac proteins. We demonstrated this methodology by visualization of the cardiac sialylated glycans in intact hearts and identification of more than 200 cardiac proteins modified with sialic acids. We further applied this methodology to investigate the sialylation in hypertrophic hearts. The imaging results revealed an increase of sialic acid biosynthesis upon the induction of cardiac hypertrophy. Quantitative proteomic analysis identified multiple sialylated proteins including neural cell adhesion molecule 1, T-kininogens, and α2-macroglobulin that were upregulated during hypertrophy. The methodology may be further extended to other types of glycosylation, as exemplified by the mucin-type O-linked glycosylation. Our results highlight the applications of metabolic glycan labeling coupled with bioorthogonal chemistry in probing the biosynthesis and function of cardiac glycome during pathophysiological responses.
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