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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]

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]

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]

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]

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]