What comes next in glycobiology.

Glycans, with their variable compositions and highly dynamic conformations, vastly expand the heterogeneity of whatever factor or cell they are attached to. These properties make them crucial contributors to biological function and organismal health and also very difficult to study. That may be changing as we look to the future of glycobiology.

Sialic acid plays a pivotal role in licensing Citrobacter rodentium's transition from the intestinal lumen to a mucosal adherent niche.

Enteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses and an established microbiota remain poorly defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium, its virulence strategy likely involves metabolic adaptation to the host's intestinal luminal environment, as a necessary precursor to reach and infect the mucosal surface. Suspecting this adaptation involved the intestinal mucus layer, we found that C. rodentium was able to catabolize sialic acid, a monosaccharide derived from mucins, and utilize it as its sole carbon source for growth. Moreover, C. rodentium also sensed and displayed chemotactic activity toward sialic acid. These activities were abolished when the nanT gene, encoding a sialic acid transporter, was deleted (ΔnanT). Correspondingly, the ΔnanT C. rodentium strain was significantly impaired in its ability to colonize the murine intestine. Intriguingly, sialic acid was also found to induce the secretion of two autotransporter proteins, Pic and EspC, which possess mucinolytic and host-adherent properties. As a result, sialic acid enhanced the ability of C. rodentium to degrade intestinal mucus (through Pic), as well as to adhere to intestinal epithelial cells (through EspC). We thus demonstrate that sialic acid, a monosaccharide constituent of the intestinal mucus layer, functions as an important nutrient and a key signal for an A/E bacterial pathogen to escape the colonic lumen and directly infect its host's intestinal mucosa.

Experimentally Determined Diagnostic Ions for Identification of Peptide Glycotopes.

The analysis of the structures of glycans present on glycoproteins is an essential component for determining glycoprotein function; however, detailed glycan structural assignment on glycopeptides from proteomics mass spectrometric data remains challenging. Glycoproteomic analysis by mass spectrometry currently can provide significant, yet incomplete, information about the glycans present, including the glycan monosaccharide composition and in some circumstances the site(s) of glycosylation. Advancements in mass spectrometric resolution, using high-mass accuracy instrumentation and tailored MS/MS fragmentation parameters, coupled with a dedicated definition of diagnostic fragmentation ions have enabled the determination of some glycan structural features, or glycotopes, expressed on glycopeptides. Here we present a collation of diagnostic glycan fragments produced by traditional positive-ion-mode reversed-phase LC-ESI MS/MS proteomic workflows and describe the specific fragmentation energy settings required to identify specific glycotopes presented on N- or O-linked glycopeptides in a typical proteomics MS/MS experiment.

The 2023 Report on the Proteome from the HUPO Human Proteome Project.

Since 2010, the Human Proteome Project (HPP), the flagship initiative of the Human Proteome Organization (HUPO), has pursued two goals: (1) to credibly identify the protein parts list and (2) to make proteomics an integral part of multiomics studies of human health and disease. The HPP relies on international collaboration, data sharing, standardized reanalysis of MS data sets by PeptideAtlas and MassIVE-KB using HPP Guidelines for quality assurance, integration and curation of MS and non-MS protein data by neXtProt, plus extensive use of antibody profiling carried out by the Human Protein Atlas. According to the neXtProt release 2023-04-18, protein expression has now been credibly detected (PE1) for 18,397 of the 19,778 neXtProt predicted proteins coded in the human genome (93%). Of these PE1 proteins, 17,453 were detected with mass spectrometry (MS) in accordance with HPP Guidelines and 944 by a variety of non-MS methods. The number of neXtProt PE2, PE3, and PE4 missing proteins now stands at 1381. Achieving the unambiguous identification of 93% of predicted proteins encoded from across all chromosomes represents remarkable experimental progress on the Human Proteome parts list. Meanwhile, there are several categories of predicted proteins that have proved resistant to detection regardless of protein-based methods used. Additionally there are some PE1-4 proteins that probably should be reclassified to PE5, specifically 21 LINC entries and ∼30 HERV entries; these are being addressed in the present year. Applying proteomics in a wide array of biological and clinical studies ensures integration with other omics platforms as reported by the Biology and Disease-driven HPP teams and the antibody and pathology resource pillars. Current progress has positioned the HPP to transition to its Grand Challenge Project focused on determining the primary function(s) of every protein itself and in networks and pathways within the context of human health and disease.

SSSMuG: Same Sample Sequential Multi-Glycomics.

The mammalian glycome is structurally complex and diverse, composed of many glycan classes such as N- and O-linked glycans, glycosaminoglycans (GAGs), glycosphingolipids (GSLs), and other distinct glycan features such as polysialic acids (PolySia), sulfation, and proteoglycan attachment stubs. Various methods are used to analyze these different components of the glycome, but they require prefractionated/partitioned samples to target each glycan class individually. To address this need for a knowledge of the relationship between the different glycan components of a biological system, we developed a sequential release workflow for analysis of multiple conjugated glycan classes (PolySia, GAGs, GSL glycans, N-glycans, and O-glycans) from the same tissue lysate, termed SSSMuG─Same Sample Sequential Multi-Glycomics. With this sequential glycan release approach, five glycan classes were characterized (or four glycan classes plus proteomics) using enzymatic or chemical release from a single sample immobilized on a polyvinylidene difluoride membrane. The various released glycan classes were then analyzed by HPLC and MS techniques using commonly available analytical setups. Compared to single glycan class release approaches, SSSMuG was able to identify more glycans and more proteins with higher-intensity analytical peaks and provide a better comparative normalization of the different glycan classes of the complex glycome. To this end, the SSSMuG technology workflow will be a foundation for a paradigm shift in the field, transforming glycoanalytics and facilitating the push toward multiglycomics and systems glycobiology.

Lectin-conjugated nanotags with high SERS stability: selective probes for glycans.

Surface-enhanced Raman scattering (SERS) nanotags functionalized with lectins as the biological recognition element can be used to target the carbohydrate portion of carbohydrate-carrying molecules (glycoconjugates). An investigation of the optical stability of such functionalized SERS nanotags is an essential initial step before future application and quantification of surface glycan biomarkers on cells and extracellular vesicles. Herein, we report an innovative approach to evaluate the SERS stability of lectin-conjugated nanotags by investigating any possible interfering lectin-lectin interactions in a mixture of different lectin-conjugated SERS nanotags, as well as an assessment of lectin-glycan interaction by mixing wheat germ agglutinin (WGA)-conjugated SERS nanotags with different glycoproteins. No lectin cross-reactivity was found in the mixture of lectin-conjugated SERS nanotags, evidenced by the constant SERS intensity. Additionally, the results showed that the lectins conjugated to SERS nanotags retain their ability to interact with glycans, as evidenced by the changes in the nanotag color and extinction spectra. Their SERS intensity remained constant as supported by finite-element method (FEM) simulation results, demonstrating a high SERS stability and selectivity of lectin-conjugated nanotags towards multiplex applications.

The 2022 Report on the Human Proteome from the HUPO Human Proteome Project.

The 2022 Metrics of the Human Proteome from the HUPO Human Proteome Project (HPP) show that protein expression has now been credibly detected (neXtProt PE1 level) for 18 407 (93.2%) of the 19 750 predicted proteins coded in the human genome, a net gain of 50 since 2021 from data sets generated around the world and reanalyzed by the HPP. Conversely, the number of neXtProt PE2, PE3, and PE4 missing proteins has been reduced by 78 from 1421 to 1343. This represents continuing experimental progress on the human proteome parts list across all the chromosomes, as well as significant reclassifications. Meanwhile, applying proteomics in a vast array of biological and clinical studies continues to yield significant findings and growing integration with other omics platforms. We present highlights from the Chromosome-Centric HPP, Biology and Disease-driven HPP, and HPP Resource Pillars, compare features of mass spectrometry and Olink and Somalogic platforms, note the emergence of translation products from ribosome profiling of small open reading frames, and discuss the launch of the initial HPP Grand Challenge Project, "A Function for Each Protein".

Mouse brain glycomics - Insights from exploring the Allen Brain Atlas and the implications for the neuroimmune brain.

The Allen Institute Mouse Brain Atlas, with visualisation using the Brain Explorer software, offers a 3-dimensional view of region-specific RNA expression of thousands of mouse genes. In this Viewpoint, we focused on the region-specific expression of genes related to cellular glycosylation, and discuss their relevance towards psychoneuroimmunology. Using specific examples, we show that the Atlas validates existing observations reported by others, identifies previously unknown potential region-specific glycan features, and highlights the need to promote collaborations between glycobiology and psychoneuroimmunology researchers.

Membrane glycome is impacted by the cell culturing mode of neuroblastoma cells with differing migration and invasion potential.

Neuroblastoma is a highly metastatic childhood cancer for which studies indicate an association between protein glycosylation and tumor behavior. However, there is a lack of detailed glycome analysis on neuroblastoma cells that have varying metastatic potential. Furthermore, the impact of the cell culturing mode, i.e. 2-dimensional (2D) versus 3-dimensional (3D) spheroids, on the membrane protein glycome is unknown. To address these gaps in knowledge, we mapped membrane protein N- and O-glycosylation of neuroblastoma cells that have lower invasive and metastatic potential (Stathmin shRNA-expressing cells, StmnSeq2SH, and StmnSeq3SH) compared with control cells (control shRNA-expressing cells, CtrlSH). We showed that the neuroblastoma cells with different migratory and invasive potential underwent drastic changes in their membrane protein N-glycosylation exclusively when cultured in 3D spheroids. We also investigated the impact of 2D and 3D cell culture methods on cellular glycosylation using the neuroblastoma cells and found the cell N-glycome was markedly impacted by the culture method, with the 2D grown cells showing an abundance of oligomannosidic glycans, whereas 3D spheroids expressed more complex type glycans on their membrane proteins. In summary, this study provides the first comprehensive protein glycome profiling of neuroblastoma cells that have varying invasiveness and migratory potential and unravels the distinct membrane glycan features of cells that are grown under 2D versus 3D culture conditions.

Long-term intrathecal administration of morphine vs. baclofen: Differences in CSF glycoconjugate profiles using multiglycomics.

Opioid use for treatment of persistent pain has increased dramatically over the past two decades, but it has not resulted in improved pain management outcomes. To understand the molecular mechanisms of opioids, molecular signatures that arise from opioid exposure are often sought after, using various analytical methods. In this study, we performed proteomics, and multiglycomics via sequential analysis of polysialic acids, glycosaminoglycans, N-glycans and O-glycans, using the same cerebral spinal fluid (CSF) sample from patients that had long-term (>2 years), intrathecal morphine or baclofen administered via an indwelling pump. Proteomics and N-glycomics signatures between the two treatment groups were highly conserved, while significant differences were observed in polysialic acid, heparan sulfate glycosaminoglycan and O-glycan profiles between the two treatment groups. This represents the first study to investigate the potential relationships between diverse CSF conjugated glycans and long-term intrathecal drug exposure. The unique changes, observed by a sequential analytical workflow, reflect previously undescribed molecular effects of opioid administration and pain management.

Cancer-derived small extracellular vesicles: emerging biomarkers and therapies for pancreatic ductal adenocarcinoma diagnosis/prognosis and treatment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers worldwide with high mortality, which is mainly due to the lack of reliable biomarkers for PDAC diagnosis/prognosis in the early stages and effective therapeutic strategies for the treatment. Cancer-derived small extracellular vesicles (sEVs), which carry various messages and signal biomolecules (e.g. RNAs, DNAs, proteins, lipids, and glycans) to constitute the key features (e.g. genetic and phenotypic status) of cancer cells, are regarded as highly competitive non-invasive biomarkers for PDAC diagnosis/prognosis. Additionally, new insights on the biogenesis and molecular functions of cancer-derived sEVs pave the way for novel therapeutic strategies based on cancer-derived sEVs for PDAC treatment such as inhibition of the formation or secretion of cancer-derived sEVs, using cancer-derived sEVs as drug carriers and for immunotherapy. This review provides a comprehensive overview of the most recent scientific and clinical research on the discovery and involvement of key molecules in cancer-derived sEVs for PDAC diagnosis/prognosis and strategies using cancer-derived sEVs for PDAC treatment. The current limitations and emerging trends toward clinical application of cancer-derived sEVs in PDAC diagnosis/prognosis and treatment have also been discussed.

Bisecting GlcNAc Protein N-Glycosylation Is Characteristic of Human Adipogenesis.

Human adipose tissue contains a major source of adipose-derived stem cells (ADSCs) that have the ability to differentiate into various cell types: in vitro, ADSCs can differentiate into mesenchymal lineages including adipocytes, while in vivo, ADSCs become mature adipocytes. Protein glycosylation has been shown to change in stem cell differentiation, and while ADSCs have been acknowledged for their therapeutic potential, little is known about protein glycosylation during human ADSC adipogenic differentiation. In the present study, the global membrane protein glycosylation of native adipocytes was compared to ADSCs from the same individuals as a model of in vivo adipogenesis. For in vitro adipogenesis, ADSCs were adipogenically differentiated in cell culture using an optimized, large-scale differentiation procedure. The membrane glycome of the differentiated ADSCs (dADSCs) was compared with mature adipocytes and the progenitor ADSCs. A total of 137 glycan structures were characterized across the three cell types using PGC-LC coupled with negative-ion electrospray ionization mass spectrometry (ESI-MS)/MS. Significantly higher levels of bisecting GlcNAc-type N-glycans were detected in mature adipocytes (32.1% of total glycans) and in in vitro dADSC progeny (1.9% of total glycans) compared to ADSCs. This was further correlated by the mRNA expression of the MGAT3 gene responsible for the enzymatic synthesis of this structural type. The bisecting GlcNAc structures were found on the majority of human native adipocyte membrane proteins, suggesting an important role in human adipocyte biology. Core fucosylation was also significantly increased during in vivo adipogenesis but did not correlate with an increase in Fut8 gene transcript. Unexpectedly, low abundance structures carrying rare ß-linked Gal-Gal termini were also detected. Overall, the N-glycan profiles of the in vitro differentiated progeny did not reflect native adipocytes, and the results show that bisecting GlcNAc structures are a characteristic feature of human adipocyte membrane protein N-glycosylation. Raw MS files are available on GlycoPOST (ID: GPST000153 https://glycopost.glycosmos.org/).

The Hitchhiker's guide to glycoproteomics.

Protein glycosylation is one of the most common post-translational modifications that are essential for cell function across all domains of life. Changes in glycosylation are considered a hallmark of many diseases, thus making glycoproteins important diagnostic and prognostic biomarker candidates and therapeutic targets. Glycoproteomics, the study of glycans and their carrier proteins in a system-wide context, is becoming a powerful tool in glycobiology that enables the functional analysis of protein glycosylation. This 'Hitchhiker's guide to glycoproteomics' is intended as a starting point for anyone who wants to explore the emerging world of glycoproteomics. The review moves from the techniques that have been developed for the characterisation of single glycoproteins to technologies that may be used for a successful complex glycoproteome characterisation. Examples of the variety of approaches, methodologies, and technologies currently used in the field are given. This review introduces the common strategies to capture glycoprotein-specific and system-wide glycoproteome data from tissues, body fluids, or cells, and a perspective on how integration into a multi-omics workflow enables a deep identification and characterisation of glycoproteins - a class of biomolecules essential in regulating cell function.

Enzymatic Azido-GalNAc-Functionalized Silk Fibroin for Click Chemistry Conjugation.

Silk is a popular protein biomaterial that has been used for various purposes such as tissue scaffolding, textiles and hydrogels. Various methods for covalent conjugation of functional molecules such as small molecule sensors and enzymes have been developed to create functionalized silk biomaterials. Here, we report a method for silk functionalization by using O-GalNAc-transferases and azide-modified UDP-GalNAc nucleotide sugar substrates to incorporate azide functional groups onto the silk fibroin protein for functionalization with cycloalkynes by click chemistry. Using ppGalNAc-T1 and T13 enzymes, we could transfer azide-modified GalNAc monosaccharides onto fibroin and as a proof of concept, conjugated a strain-alkyne-functionalized Cy5 fluorophore to produce a Cy5-conjugated fibroin hydrogel. This facile azido functionalization of the silk has the potential for attachment of any cycloalkyne moiety.

Altered N-linked glycosylation in endometrial cancer.

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.

Light-Emitting Diode Excitation for Upconversion Microscopy: A Quantitative Assessment.

Lanthanide-based upconversion nanoparticles (UCNPs) generally require high power laser excitation. Here, we report wide-field upconversion microscopy at single-nanoparticle sensitivity using incoherent excitation of a 970 nm light-emitting diode (LED). We show that due to its broad emission spectrum, LED excitation is about 3 times less effective for UCNPs and generates high background compared to laser illumination. To counter this, we use time-gated luminescence detection to eliminate the residual background from the LED source, so that individual UCNPs with high sensitizer (Yb3+) doping and inert shell protection become clearly identified under LED excitation at 1.18 W cm-2, as confirmed by correlated electron microscopy images. Hydrophilic UCNPs are obtained by polysaccharide coating via a facile ligand exchange protocol to demonstrate imaging of cellular uptake using LED excitation. These results suggest a viable approach to bypassing the limitations associated with high-power lasers when applying UCNPs and upconversion microscopy to life science research.

Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells.

Colonization of the oropharynx is the initial step in Group A Streptococcus (GAS) pharyngeal infection. We have previously reported that the highly virulent M1T1 GAS clone attaches to oral epithelial cells via M1 protein interaction with blood group antigen carbohydrate structures. Here, we have identified that colonization of human oral epithelial cells by GAS serotypes M3 and M12 is mediated by human blood group antigens [ABO(H)] and Lewis (Le) antigen expression. Removal of linkage-specific fucose, galactose, N-acetylgalactosamine, and sialic acid modulated GAS colonization, dependent on host ABO(H) blood group and Le expression profile. Furthermore, N-linked glycans from human salivary glycoproteins, when released and purified, were potent inhibitors of M1, M3, and M12 GAS colonization ex vivo. These data highlight the important role played by human protein glycosylation patterns in GAS attachment to oral epithelial cell surfaces.-De Oliveira, D. M. P., Everest-Dass, A., Hartley-Tassell, L., Day, C. J., Indraratna, A., Brouwer, S., Cleary, A., Kautto, L., Gorman, J., Packer, N. H., Jennings, M. P., Walker, M. J., Sanderson-Smith, M. L. Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells.

Glycomics@ExPASy: Bridging the Gap.

Glycomics@ExPASy (https://www.expasy.org/glycomics) is the glycomics tab of ExPASy, the server of SIB Swiss Institute of Bioinformatics. It was created in 2016 to centralize web-based glycoinformatics resources developed within an international network of glycoscientists. The hosted collection currently includes mainly databases and tools created and maintained at SIB but also links to a range of reference resources popular in the glycomics community. The philosophy of our toolbox is that it should be {glycoscientist AND protein scientist}-friendly with the aim of (1) popularizing the use of bioinformatics in glycobiology and (2) emphasizing the relationship between glycobiology and protein-oriented bioinformatics resources. The scarcity of data bridging these two disciplines led us to design tools as interactive as possible based on database connectivity to facilitate data exploration and support hypothesis building. Glycomics@ExPASy was designed, and is developed, with a long-term vision in close collaboration with glycoscientists to meet as closely as possible the growing needs of the community for glycoinformatics.

MALDI Mass Spectrometry Imaging of Early- and Late-Stage Serous Ovarian Cancer Tissue Reveals Stage-Specific N-Glycans.

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex)6 + (Man)3 (GlcNAc)2 , and the complex neutral structure, (Hex)2 (HexNAc)2 (Deoxyhexose)1 + (Man)3 (GlcNAc)2 . The distribution of these markers is evaluated using a tissue microarray of early- and late-stage patients.

Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.