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Proteins are ubiquitously modified with glycans of varied chemical structures through distinct glycosidic linkages, making the landscape of protein glycosylation challenging to map. Profiling of intact glycopeptides with mass spectrometry (MS) has recently emerged as a powerful tool for revealing matched information of the glycosylation sites and attached glycans (i.e. intact glycosites), but is largely limited to individual glycosylation types. Herein, we describe Click‐iG, which integrates metabolic labeling of glycans with clickable unnatural sugars, an optimized MS method, and a tailored version of pGlyco3 software to enable simultaneous enrichment and profiling of three types of intact glycopeptides: N‐linked, mucin‐type O‐linked, and O‐GlcNAcylated glycopeptides. We demonstrate the utility of Click‐iG by the identification of thousands of intact glycosites in cell lines and living mice. From the mouse lung, heart, and spleen, a total of 2053 intact N‐glycosites, 262 intact O‐GalNAc glycosites, and 1947 O‐GlcNAcylation sites were identified. Click‐iG‐enabled comprehensive coverage of the protein glycosylation landscape lays the foundation for interrogating crosstalk between different glycosylation pathways. [ABSTRACT FROM AUTHOR]

Clickable unnatural sugars have been widely used in studying glycosylation in living systems via the metabolic glycan labelling (MGL) strategy. Partial protection of unnatural sugars by 1,6‐di‐O‐acylation increases the labelling efficiency while avoiding the non‐specific S‐glyco‐modification. Herein, we report the facile synthesis of a series of clickable unnatural sugars in both the unprotected and 1,6‐di‐O‐acylated forms at the ten‐gram scale. By evaluation of the labelling specificity, efficiency, and biocompatibility of various 1,6‐di‐O‐acylated sugars for MGL in cell lines and living mice, we demonstrate that 1,6‐di‐O‐propionylated unnatural sugars are optimal chemical reporters for glycan labelling. The synthetic routes developed in this work should facilitate the widespread use of MGL with no artificial S‐glyco‐modification for investigating the functional roles of glycans. [ABSTRACT FROM AUTHOR]

Protein O‐GlcNAcylation is a ubiquitous posttranslational modification occurring both in animals and plants. While thousands of O‐GlcNAcylated proteins have been identified in animals, the plant O‐GlcNAcylated proteome remains poorly studied. Herein we report the development of a chemoproteomic strategy for profiling of O‐GlcNAcylated proteins in Arabidopsis based on the metabolic glycan labeling (MGL) method. We first demonstrated that both N‐azidoacetylglucosamine (GlcNAz) and N‐azidoacetylgalactosamine (GalNAz) can metabolically label O‐GlcNAc with azides in Arabidopsis seedlings. Arabidopsis UDP‐galactose 4‐epimerases were found to interconvert UDP‐GalNAz and UDP‐GlcNAz, supporting the existence of a GalNAc metabolism pathway. By tagging the azide‐incorporated O‐GlcNAc with alkyne‐biotin via click chemistry, the O‐GlcNAcylated proteins were enriched and analyzed by mass spectrometry. We identified 645 candidate O‐GlcNAcylated proteins in Arabidopsis seedlings, of which 592 were newly identified. The identified O‐GlcNAcylated proteins were enriched in various plant‐specific processes such as hormone responses. By co‐expression of a selected list of the identified proteins with SECRET AGENT, the Arabidopsis O‐GlcNAc transferase, we validated that the MGL‐identified proteins were O‐GlcNAc‐modified. Our work establishes a powerful tool for profiling plant O‐GlcNAylation and provides an invaluable resource for investigating the functional role of O‐GlcNAc in Arabidopsis. [ABSTRACT FROM AUTHOR]

We report here the development of clickable and highly near‐infrared (NIR) fluorescent lanthanide (Ln) complexes for bioorthogonal labeling of biomolecules. These azide‐ or alkyne‐functionalized Ln complexes are hydrophilic and fluorogenic, exhibiting a strong increase of NIR fluorescence upon conjugation with biomolecules. Metabolic labeling of biomolecules with azide or alkyne, followed by click labeling with the Ln complexes, enables NIR fluorescence (NIRF) imaging of DNA, RNA, proteins, and glycans in cells. Furthermore, multicolor imaging is performed by combining click‐labeling with the Ln complexes and immunostaining. In addition, the Ln complexes is compatible with click‐expansion microscopy (click‐ExM), which enables high‐resolution NIRF imaging of cellular glycoproteins. Finally, the Ln complexes can be used for time‐of‐flight secondary‐ion mass spectrometry (ToF‐SIMS) imaging, thus achieving the first example of dual‐modal imaging combining NIRF and SIMS microscopies. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: The photocaging strategy has been widely used for activating biomolecules and hence regulating the underlying biological processes with high temporal and spatial resolution. Herein, we integrate this strategy into metabolic glycan labeling, which enables optically‐controlled metabolic labeling of cell‐surface sialoglycans with azides. N‐Azidoacetylmannosamine (ManNAz) is caged with a 4,5‐dimethoxy‐2‐nitrobenzyl (DMNB) group at the anomeric hydroxyl group to block its cellular metabolism. After feeding to the cells, irradiation with 365 nm UV light or 405 nm visible light cleaves the photocage and generates ManNAz within the cells. ManNAz is metabolically converted to azido sialic acid, which is then incorporated into cell‐surface sialoglycans. We demonstrated spatial and temporal control of this strategy by specifically labeling sialoglycans on a single cell and tracking the metabolism of ManNAz based on light‐triggered pulse‐chase labeling, respectively. The light‐controlled metabolic glycan labeling strategy should facilitate probing of glycosylation in complex biological systems with high spatiotemporal precision. [ABSTRACT FROM AUTHOR]

A chemical approach was developed for identifying cell-surface markers for primary neural stem cells (NSCs). Using an in vitro coculture system of primary NSCs combined with metabolic labeling of sialoglycans with bioorthogonal functional groups, we selectively enriched and identified a list of cell-surface sialoglycoproteins that were more abundantly expressed in neural stem and progenitor cells. [ABSTRACT FROM AUTHOR]

Abstract: The unexpected, non‐enzymatic S‐glycosylation of cysteine residues in various proteins by per‐O‐acetylated monosaccharides is described. This artificial S‐glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per‐O‐acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O‐GlcNAcylated proteins and O‐GlcNAc sites in HeLa cells has been assembled by using N‐azidoacetylgalactosamine (GalNAz). [ABSTRACT FROM AUTHOR]

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N-azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N-acetylglucosamine (GlcNAc) and incorporated into N-linked glycans, and possibly intracellular O-GlcNAc. Click-labeling with fluorescent probes enables visualization of newly synthesized N-linked glycans. N-glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N-glycosylation is regulated in a developmental stage-dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N-linked glycosylation in plants. [ABSTRACT FROM AUTHOR]

We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium-mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N-(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell-surface glycans. This conversion chemically mimics the enzymatic de-N-acetylation of N-acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell-surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell-surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids. [ABSTRACT FROM AUTHOR]

We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium-mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N-(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell-surface glycans. This conversion chemically mimics the enzymatic de-N-acetylation of N-acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell-surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell-surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids. [ABSTRACT FROM AUTHOR]

Although it has been well known that dynamic changes in glycosylation are associated with tumor progression, it remains challenging to selectively visualize the cancer glycome in vivo. Herein, a strategy for the targeted imaging of tumor-associated glycans by using ligand-targeted liposomes encapsulating azidosugars is described. The intravenously injected liposomal nanoparticles selectively bound to the cancer-cell-specific receptors and installed azides into the melanoma glycans in a xenograft mouse model in a tissue-specific manner. Subsequently, a copper-free click reaction was performed in vivo to chemoselectively conjugate the azides with a near-infrared fluorescent dye. The glycosylation dynamics during tumor growth were monitored by in vivo fluorescence imaging. Furthermore, the newly synthesized sialylated glycoproteins were enriched during tumor growth and identified by glycoproteomics. Compared with the labeling methods using free azidosugars, this method offers improved labeling efficiency and high specificity and should facilitate the elucidation of the functional role of glycans in cancer biology. [ABSTRACT FROM AUTHOR]

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman-silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label-free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state-of-the-art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non-invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live-cell imaging. [ABSTRACT FROM AUTHOR]

Direct detection of glycans on live cells using surface‐enhanced Raman scattering (SERS) has been shown. A bioorthogonal Raman reporter was directly installed onto the monosaccharide analogs. Once metabolically incorporated into cell surface glycans, the Raman reporter was detected using SERS (see picture). [ABSTRACT FROM AUTHOR]

Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per-O-acetylated, which, however, can induce a long-overlooked side reaction, non-enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N-azidoacetylgalactosamine (GalNAz) as next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O-propionylated GalNAz (1,3-Pr2GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG. Per-O-acetylated unnatural monosaccharides are popular tools for glycan labeling in live cells but can undergo unwanted side reactions with cysteines. Here, the authors develop unnatural sugars in a partially esterified form that are inert towards cysteines, and use them to probe O-GlcNAcylation in mESCs. [ABSTRACT FROM AUTHOR]

Raman detection of glycans on surfaces of live cells has been achieved, as described in the Communication by Z.‐Q. Tian, X. Chen, and co‐workers on page 7266 ff. A bioorthogonal Raman reporter (for alkyne, azide, and nitrile groups, or carbon–deuterium bonds) was installed onto the monosaccharide analogues, which were metabolically incorporated into cell surface glycans. The Raman reporter is detected using surface‐enhanced Raman scattering with the aid of gold nanoparticles. [ABSTRACT FROM AUTHOR]

Highlights: [•] General methodology for cell-selective metabolic labeling of biomolecules. [•] Enzymatic engineering for cell-selective metabolic protein labeling. [•] Caging strategy for cell-selective metabolic glycan labeling. [•] Liposomal delivery strategy for cell-selective metabolic glycan labeling. [ABSTRACT FROM AUTHOR]

dendritic cell (DC)-specific intracellular adhesion molecule-3grabbing nonintegrin (DC-SIGN) is a receptor found on DCs that recognizesantigens bearing mannose-rich or fucosylated glycans, including LewisX (LeX). Here, we report the fabrication of magnetic nanoparticlescoated with multivalent LeXglycans using Cu (I)-catalyzedazide–alkyne cycloaddition. The resulting nanoparticles areselective and biocompatible, serving as a highly efficient tool forDC detection and enrichment. [ABSTRACT FROM AUTHOR]