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The Human Proteome Project (HPP), the flagship initiative of the Human Proteome Organization (HUPO), has pursued two goals: (1) to credibly identify at least one isoform of every protein-coding gene and (2) to make proteomics an integral part of multiomics studies of human health and disease. The past year has seen major transitions for the HPP. neXtProt was retired as the official HPP knowledge base, UniProtKB became the reference proteome knowledge base, and Ensembl-GENCODE provides the reference protein target list. A function evidence FE1-5 scoring system has been developed for functional annotation of proteins, parallel to the PE1-5 UniProtKB/neXtProt scheme for evidence of protein expression. This report includes updates from neXtProt (version 2023-09) and UniProtKB release 2024_04, with protein expression detected (PE1) for 18138 of the 19411 GENCODE protein-coding genes (93%). The number of non-PE1 proteins ("missing proteins") is now 1273. The transition to GENCODE is a net reduction of 367 proteins (19,411 PE1-5 instead of 19,778 PE1-4 last year in neXtProt). We include reports from the Biology and Disease-driven HPP, the Human Protein Atlas, and the HPP Grand Challenge Project. We expect the new Functional Evidence FE1-5 scheme to energize the Grand Challenge Project for functional annotation of human proteins throughout the global proteomics community, including π-HuB in China.

Metabolic diseases such as obesity and type 2 diabetes are marked by insulin resistance 1,2 . Cells within the arcuate nucleus of the hypothalamus (ARC), which are crucial for regulating metabolism, become insulin resistant during the progression of metabolic disease 3-8 , but these mechanisms are not fully understood. Here we investigated the role of a specialized chondroitin sulfate proteoglycan extracellular matrix, termed a perineuronal net, which surrounds ARC neurons. In metabolic disease, the perineuronal net of the ARC becomes augmented and remodelled, driving insulin resistance and metabolic dysfunction. Disruption of the perineuronal net in obese mice, either enzymatically or with small molecules, improves insulin access to the brain, reversing neuronal insulin resistance and enhancing metabolic health. Our findings identify ARC extracellular matrix remodelling as a fundamental mechanism driving metabolic diseases., (© 2024. The Author(s).)

Synthetic vascular grafts are used to bypass significant arterial blockage when native blood vessels are unsuitable, yet their propensity to fail due to poor blood compatibility and progressive graft stenosis remains an intractable challenge. Perlecan is the major heparan sulfate (HS) proteoglycan in the blood vessel wall with an inherent ability to regulate vascular cell activities associated with these major graft failure modes. Here the ability of the engineered form of perlecan domain V (rDV) to bind angiogenic growth factors is tuned and endothelial cell proliferation via the composition of its glycosaminoglycan (GAG) chain is supported. It is shown that the HS on rDV supports angiogenic growth factor signaling, including fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF)165, while both HS and chondroitin sulfate on rDV are involved in VEGF189 signaling. It is also shown that physisorption of rDV on emerging electrospun silk fibroin vascular grafts promotes endothelialization and patency in a murine arterial interposition model, compared to the silk grafts alone. Together, this study demonstrates the potential of rDV as a tunable, angiogenic biomaterial coating that both potentiates growth factors and regulates endothelial cells., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Reduced responsiveness of precursor B-acute lymphoblastic leukemia (BCP-ALL) to chemotherapy can be first detected in the form of minimal residual disease leukemia cells that persist after 28 days of initial treatment. The ability of these cells to resist chemotherapy is partly due to the microenvironment of the bone marrow, which promotes leukemia cell growth and provides protection, particularly under these conditions of stress. It is unknown if and how the glycocalyx of such cells is remodelled during the development of tolerance to drug treatment, even though glycosylation is the most abundant cell surface post-translational modification present on the plasma membrane. To investigate this, we performed omics analysis of BCP-ALL cells that survived a 30-day vincristine chemotherapy treatment while in co-culture with bone marrow stromal cells. Proteomics showed decreased levels of some metabolic enzymes. Overall glycocalyx changes included a shift from Core-2 to less complex Core-1 O-glycans, and reduced overall sialylation, with a shift from α2-6 to α2-3 linked Neu5Ac. Interestingly, there was a clear increase in bisecting complex N-glycans with a concomitant increased mRNA expression of MGAT3 , the only enzyme known to form bisecting N-glycans. These small but reproducible quantitative differences suggest that individual glycoproteins become differentially glycosylated. Glycoproteomics confirmed glycosite-specific modulation of cell surface and lysosomal proteins in drug-tolerant BCP-ALL cells, including HLA-DRA, CD38, LAMP1 and PPT1. We conclude that drug-tolerant persister leukemia cells that grow under continuous chemotherapy stress have characteristic glycotraits that correlate with and perhaps contribute to their ability to survive and could be tested as neoantigens in drug-resistant leukemia.

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.

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., Competing Interests: Declaration of interests B.G.D. is a shareholder of SugaROx and holds various patents that seek to exploit the function of glycosylation and that would afford him royalties upon exploitation. G.A.R. is co-founder of Galtec SAS and co-inventor on a family of US and European patents associated with therapeutic and diagnostic applications of galectins in cancer and autoimmune and inflammatory conditions. P.H.S. has a significant financial interest in GlycoUniverse GmbH&CoKG (commercializing the automated synthesis technology and services related to it) and Tacalyx GmbH (developing anti-glycan antibodies to treat cancer patients) as well as advisory roles with both companies. He holds a host of patents in the area of synthetic carbohydrates and their use as vaccines and for the generation of therapeutic antibodies., (Copyright © 2024. Published by Elsevier Inc.)

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 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.

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.

The structures and functions of organelles in cells depend on each other but have not been systematically explored. We established stable knockout cell lines of peroxisomal, Golgi and endoplasmic reticulum genes identified in a whole-genome CRISPR knockout screen for inducers of mitochondrial biogenesis stress, showing that defects in peroxisome, Golgi and endoplasmic reticulum metabolism disrupt mitochondrial structure and function. Our quantitative total-organelle profiling approach for focussed ion beam scanning electron microscopy revealed in unprecedented detail that specific organelle dysfunctions precipitate multi-organelle biogenesis defects, impair mitochondrial morphology and reduce respiration. Multi-omics profiling showed a unified proteome response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised, and that the resulting mitochondrial dysfunction can be rescued with precursors for ether-glycerophospholipid metabolic pathways. This work defines metabolic and morphological interactions between organelles and how their perturbation can cause disease., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Growth factors are key molecules involved in angiogenesis, a process critical for tissue repair and regeneration. Despite the potential of growth factor delivery to stimulate angiogenesis, limited clinical success has been achieved with this approach. Growth factors interact with the extracellular matrix (ECM), and particularly heparan sulphate (HS), to bind and potentiate their signalling. Here we show that engineered short forms of perlecan, the major HS proteoglycan of the vascular ECM, bind and signal angiogenic growth factors, including fibroblast growth factor 2 and vascular endothelial growth factor-A. We also show that engineered short forms of perlecan delivered in porous chitosan biomaterial scaffolds promote angiogenesis in a rat full thickness dermal wound model, with the fusion of perlecan domains I and V leading to superior vascularisation compared to native endothelial perlecan or chitosan scaffolds alone. Together, this study demonstrates the potential of engineered short forms of perlecan delivered in chitosan scaffolds as next generation angiogenic therapies which exert biological activity via the potentiation of growth factors., Competing Interests: Declaration of Competing Interest The authors declare no competing interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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 (Δ nanT ). Correspondingly, the Δ nanT 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.

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".

Two molecular cytology approaches, (i) time-gated immunoluminescence assay (TGiA) and (ii) Raman-active immunolabeling assay (RiA), have been developed to detect prostate cancer (PCa) cells in urine from five prostate cancer patients. For TGiA, PCa cells stained by a biocompatible europium chelate antibody-conjugated probe were quantitated by automated time-gated microscopy (OSAM). For RiA, PCa cells labeled by antibody-conjugated Raman probe were detected by Raman spectrometer. TGiA and RiA were first optimized by the detection of PCa cultured cells (DU145) spiked into control urine, with TGiA-OSAM showing single-cell PCa detection sensitivity, while RiA had a limit of detection of 4-10 cells/mL. Blinded analysis of each patient urine sample, using MIL-38 antibody specific for PCa cells, was performed using both assays in parallel with control urine. Both assays detected very low abundance PCa cells in patient urine (3-20 PCa cells per mL by TGiA, 4-13 cells/mL by RiA). The normalized mean of the detected PCa cells per 1 ml of urine was plotted against the clinical data including prostate specific antigen (PSA) level and Clinical Risk Assessment for each patient. Both cell detection assays showed correlation with PSA in the high risk patients but aligned with the Clinical Assessment rather than with PSA levels of the low/intermediate risk patients. Despite the limited available urine samples of PCa patients, the data presented in this proof-of-principle work is promising for the development of highly sensitive diagnostic urine tests for PCa., (© 2022. Crown.)

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., (© 2022. The Author(s).)

Circulating pancreatic ductal adenocarcinoma (PDAC) derived small extracellular vesicles (sEVs) are nano-sized membranous vesicles secreted from PDAC cells and released into surrounding body fluids, such as blood. The use of plasma-derived sEVs for cancer diagnosis is particularly appealing in biomedical research because the sEVs reflect some key features ( e.g. genetic and phenotypic status) related to the organs from which they originate. For example, the surface membrane proteins and their expression level on sEVs were reported to be related to the presence and progression of PDAC. However, difficulty in sEVs isolation and lack of ultrasensitive assays for simultaneous analysis of multiple protein biomarkers on patient plasma-derived sEVs hinder their application in the clinic. In our previous study, we have demonstrated the application of magnetic beads (MBs) and surface-enhanced Raman scattering (SERS) assay for phenotypic analysis of cancer cells-derived sEVs using different cell lines. To further demonstrate the clinical application of the proposed assay, we have profiled the sEVs' phenotypes (relative expression of biomarker Glypican 1, EpCAM and CD44V6) of healthy donors and PDAC patients to enable simultaneous detection of multiple surface membrane proteins on plasma-derived sEVs. We discovered that the PDAC sEVs' phenotype signatures had high accuracy for PDAC diagnosis (100%) and showed strong correlation with cancer stages, which were further validated by the imaging techniques ( e.g. computerized tomography and magnetic resonance imaging) and also the correlation of cancer stages with CA19-9 (gold standard biomarker) and the sEVs' phenotype signatures. The present proof-of-concept study thus provides an initial investigation of using the proposed SERS assay for PDAC diagnosis and early cancer stage prediction in the clinic.

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., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Surface modification and functionalization is typically required to engineer upconversion nanoparticles (UCNPs) for biosensing and bioimaging applications. Nevertheless, despite various antibody conjugation methods having been applied to UCNPs, no consensus has been reached on the best choice, as the results from individual studies are largely unable to be compared due to inadequate assessment of the properties of the conjugated products. Here, we introduce a systematic approach to quantitatively evaluate the biological activity of antibody-conjugated UCNPs. We determine that the optimal antibody conjugation efficiency to our colominic acid polysaccharide-coated UCNPs via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS) coupling is approximately 70%, corresponding to 16 antibodies per nanoparticle of 63 nm hydrodynamic diameter, with on average 12 of the 16 antibodies maintaining their affinity to the target antigens. The binding ability of the antibody-conjugated UCNPs to the antigen was well preserved, as verified by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and cellular imaging. This is the first study to quantitate the active antibody binding capacity of polysaccharide coated UCNP nanoparticles, offering a practical guideline for benchmarking functionalised UCNPs in future studies., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glycosylation is arguably the most important functional post-translational modification in brain cells and abnormal cell surface glycan expression has been associated with neurological diseases and brain cancers. In this study we developed a novel method for uptake of fluorescent nanodiamonds (FND), carbon-based nanoparticles with low toxicity and easily modifiable surfaces, into brain cell subtypes by targeting their glycan receptors with carbohydrate-binding lectins. Lectins facilitated uptake of 120 nm FND with nitrogen-vacancy centers in three types of brain cells - U87-MG astrocytes, PC12 neurons and BV-2 microglia cells. The nanodiamond/lectin complexes used in this study target glycans that have been described to be altered in brain diseases including sialic acid glycans via wheat ( Triticum aestivum ) germ agglutinin (WGA), high mannose glycans via tomato ( Lycopersicon esculentum ) lectin (TL) and core fucosylated glycans via Aleuria aurantia lectin (AAL). The lectin conjugated nanodiamonds were taken up differently by the various brain cell types with fucose binding AAL/FNDs taken up preferentially by glioblastoma phenotype astrocyte cells (U87-MG), sialic acid binding WGA/FNDs by neuronal phenotype cells (PC12) and high mannose binding TL/FNDs by microglial cells (BV-2). With increasing recognition of glycans having a role in many diseases, the lectin bioconjugated nanodiamonds developed here are well suited for further investigation into theranostic applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (This journal is © The Royal Society of Chemistry.)

The present chapter focuses on the interactive and explorative aspects of bioinformatics resources that have been recently released in glycobiology. The comparative analysis of data in a field where knowledge is scattered, incomplete, and disconnected from main biology requires efficient visualization, integration, and interactive tools that are currently only partially implemented. This overview highlights converging efforts toward building a consistent picture of protein glycosylation., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics., (© 2021. The Author(s).)

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed-lineage leukemia gene rearrangement (MLL-r) is a poor-prognosis subtype for which additional therapeutic targets are urgently needed. Currently no multi- omics data set for primary MLL r patient cells exists that integrates transcriptomics, proteomics and glycomics to gain an inclusive picture of theranostic targets. Methods: We have integrated transcriptomics, proteomics and glycomics to i) obtain the first inclusive picture of primary patient BCP-ALL cells and identify molecular signatures that distinguish leukemic from normal precursor B-cells and ii) better understand the benefits and limitations of the applied technologies to deliver deep molecular sequence data across major cellular biopolymers. Results: MLL-r cells feature an extensive remodeling of their glycocalyx, with increased levels of Core 2-type O-glycans and complex N-glycans as well as significant changes in sialylation and fucosylation. Notably, glycosaminoglycan remodeling from chondroitin sulfate to heparan sulfate was observed. A survival screen, to determine if glycan remodeling enzymes are redundant, identified MGAT1 and NGLY1, essential components of the N-glycosylation/degradation pathway, as highly relevant within this in vitro screening. OGT and OGA, unique enzymes that regulate intracellular O-GlcNAcylation, were also indispensable. Transcriptomics and proteomics further identified Fes and GALNT7-mediated glycosylation as possible therapeutic targets. While there is overall good correlation between transcriptomics and proteomics data, we demonstrate that a systematic combined multi- omics approach delivers important diagnostic information that is missed when applying a single omics technology. Conclusions: Apart from confirming well-known MLL-r BCP-ALL glycoprotein markers, our integrated multi- omics workflow discovered previously unidentified diagnostic/therapeutic protein targets., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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., (© 2021 The Author(s).)

The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG's glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies.

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.

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.

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 β-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/).