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It is well established that cell surface glycans play a vital role in biological processes and their altered form can lead to carcinogenesis. Mass spectrometry–based techniques have become prominent for analysing N-linked glycans, for example using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Additionally, MALDI MS can be used to spatially map N-linked glycans directly from cancer tissue using a technique termed MALDI MS imaging (MALDI MSI). This powerful technique combines mass spectrometry and histology to visualise the spatial distribution of N-linked glycans on a single tissue section. Here, we performed N-glycan MALDI MSI on six endometrial cancer (EC) formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) consisting of eight EC patients with lymph node metastasis (LNM) and twenty without LNM. By doing so, several putative N-linked glycan compositions were detected that could significantly distinguish normal from cancerous endometrium. Furthermore, a complex core-fucosylated N-linked glycan was detected that could discriminate a primary tumour with and without LNM. Structural identification of these putative N-linked glycans was performed using porous graphitized carbon liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS). Overall, we observed higher abundance of oligomannose glycans in tumour compared to normal regions with AUC ranging from 0.85–0.99, and lower abundance of complex N-linked glycans with AUC ranges from 0.03–0.28. A comparison of N-linked glycans between primary tumours with and without LNM indicated a reduced abundance of a complex core-fucosylated N-glycan (Hex)2(HexNAc)2(Deoxyhexose)1+(Man)3(GlcNAc)2, in primary tumour with associated lymph node metastasis. In summary, N-linked glycan MALDI MSI can be used to differentiate cancerous endometrium from normal, and endometrial cancer with LNM from endometrial cancer without. [ABSTRACT FROM AUTHOR]

Cells are covered with glycans. The expression and distribution of specific glycans on the surface of a cell are important for various cellular functions. Imaging these glycans is essential to aid elucidation of their biological roles. Here, utilizing methods of direct fluorescent glycan imaging, in which fluorescent sialic acids are directly incorporated into substrate glycans via recombinant sialyltranferases, we report the differential distribution of N- and O-glycans and variable expression of sialyl-T antigen on HeLa cells. While the expression of N-glycans tends to be more peripheral at positions where cell–cell interaction occurs, O-glycan expression is more granular but relatively evenly distributed on positive cells. While N-glycans are expressed on all cells, sialyl-T antigen expression exhibits a wide spectrum of variation with some cells being strongly positive and some cells being almost completely negative. The differential distribution of N- and O-glycans on cell surface reflects their distinctive roles in cell biology. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans. [ABSTRACT FROM AUTHOR]

Here, we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of matrix-assisted laser desorption ionization (MALDI) N-glycan mass spectrometry imaging (MSI) and spatially resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, thus directly linking glycan imaging with intact glycopeptide identification. In total, our spatially resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slices and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation. [Display omitted] • N-glycan MALDI-MSI revealed a sialylated glycan as upregulated in canine glioma • Spatial glycoproteomics identified haptoglobin as the underlying glycoprotein • Manual assignment of glycopeptides uncovered a diverse array of glycosylation • Observed significant glycopeptide changes between tumor and benign regions Malaker et al. use a multi-omics approach, including MALDI-MSI and glycoproteomics, to understand the spatial distribution of glycans and their associated glycoproteins in canine glioma. [ABSTRACT FROM AUTHOR]

As one of the major types of biomacromolecules in the cell, glycans play essential functional roles in various biological processes. Compared with proteins and nucleic acids, the analysis of glycans in situ has been more challenging. Herein we review recent advances in the development of methods and strategies for labeling, imaging, and profiling of glycans in cells and in vivo. Cellular glycans can be labeled by affinity-based probes, including lectin and antibody conjugates, direct chemical modification, metabolic glycan labeling, and chemoenzymatic labeling. These methods have been applied to label glycans with fluorophores, which enables the visualization and tracking of glycans in cells, tissues, and living organisms. Alternatively, labeling glycans with affinity tags has enabled the enrichment of glycoproteins for glycoproteomic profiling. Built on the glycan labeling methods, strategies enabling cell-selective and tissue-specific glycan labeling and protein-specific glycan imaging have been developed. With these methods and strategies, researchers are now better poised than ever to dissect the biological function of glycans in physiological or pathological contexts.

Summary O -GlcNAcylation is a reversible serine/threonine glycosylation for regulating protein activity and availability inside cells. In a given protein, O -GlcNAcylated and unoccupied O -linked β- N -acetylglucosamine (O -GlcNAc) sites are referred to as closed and open sites, respectively. The balance between open and closed sites is believed to be dynamically regulated. In this report, closed sites are detected using in vitro incorporation of GalNAz by B3GALNT2, and open sites are detected by in vitro incorporation of GlcNAz by O -GlcNAc transferase (OGT), via click chemistry. For assessing total O -GlcNAc sites, a sample is O -GlcNAcylated in vitro by OGT before detecting by B3GALNT2. The methods are demonstrated on purified recombinant proteins including CK2, AKT1, and PFKFB3, and cellular extracts of HEK cells. Through O -GlcNAc imaging, the modification degree of O -GlcNAc in nuclei of Chinese hamster ovary cells was estimated. The detection and imaging of both open and closed O -GlcNAc sites provide a systematic approach to study this important post-translational modification. Graphical Abstract Highlights • A highly specific chemo-enzymatic method for probing O -GlcNAc is provided • A chemo-enzymatic method for probing unmodified O -GlcNAc sites is provided • Imaging of both O -GlcNAc and unmodified O -GlcNAc sites in cells is achieved • Evidence that PFKFB3 is a target for O -GlcNAcylation is shown Wu et al. describe a highly specific approach to detect/image O -GlcNAc as well as potential sites for O -GlcNAc modification on biological samples using recombinant glycosyltransferases and chemical probes. The method can be used to quickly identify proteins that are targets for O -GlcNAc modification. [ABSTRACT FROM AUTHOR]

Thousands of intracellular proteins are post-translationally modified with O-GlcNAc, and O-GlcNAcylation impacts the function of modified proteins and mediates diverse biological processes. However, the ubiquity of this important glycosylation makes it highly challenging to probe the O-GlcNAcylation state of a specific protein at the cellular level. Herein, we report the development of a FLIM-FRET-based strategy, which exploits the spatial proximity of the O-GlcNAc moiety and the attaching protein, for protein-specific imaging of O-GlcNAcylation in single cells. We demonstrated this strategy by imaging the O-GlcNAcylation state of tau and β-catenin inside the cells. Furthermore, the changes in tau O-GlcNAcylation were monitored when the overall cellular O-GlcNAc was pharmacologically altered by using the OGT and OGA inhibitors. We envision that the FLIM-FRET strategy will be broadly applicable to probe the O-GlcNAcylation state of various proteins in the cells., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)