LectoScape: A Highly Multiplexed Imaging Platform for Glycome Analysis and Biomedical Diagnosis.

Glycosylation, a fundamental biological process, involves the attachment of glycans to proteins, lipids, and RNA, and it plays a crucial role in various biological pathways. It is of great significance to obtain the precise spatial distribution of glycosylation modifications at the cellular and tissue levels. Here, we introduce LectoScape, an innovative method enabling detailed imaging of tissue glycomes with up to 1 µm resolution through image mass cytometry (IMC). This method utilizes 12 distinct, nonoverlapping lectins selected via microarray technology, enabling the multiplexed detection of a wide array of glycans. Furthermore, we developed an efficient labeling strategy for these lectins. Crucially, our approach facilitates the concurrent imaging of diverse glycan motifs, including N-glycan and O-glycan, surpassing the capabilities of existing technologies. Using LectoScape, we have successfully delineated unique glycan structures in various cell types, enhancing our understanding of the glycan distribution across human tissues. Our method has identified specific glycan markers, such as α2,3-sialylated Galß1, 3GalNAc in O-glycan, and terminal GalNAc, as diagnostic indicators for cervical intraepithelial neoplasia. This highlights the potential of LectoScape in cancer diagnostics through the detection of abnormal glycosylation patterns.

O-GlycoIsoQuant: A Novel O-Glycome Quantitative Approach through Superbase Release and Isotopic Girard's P Labeling.

Quantitative glycosylation analysis serves as an effective tool for detecting changes in glycosylation patterns in cancer and various diseases. However, compared with N-glycans, O-glycans present challenges in both qualitative and quantitative mass spectrometry analysis due to their low abundance, ease of peeling, lack of a universal enzyme, and difficult accessibility. To address this challenge, we developed O-GlycoIsoQuant, a novel O-glycome quantitative approach utilizing superbase release and isotopic Girard's P labeling. This method facilitates rapid and efficient nonreducing ß-elimination to dissociate O-glycans from proteins using the organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), combined with light and heavy isotopic Girard's reagent P (GP) labeling for relative quantification of O-glycans by mass spectrometry. Employing this method, labeled O-glycans exhibit a double peak with a mass difference of 5 Da, suitable for stable relative quantification. The O-GlycoIsoQuant method is characterized by its high labeling efficiency, excellent reproducibility (CV < 20%), and good linearity (R2 > 0.99), across a dynamic range spanning a 100-fold range. This method was applied to various complex sample types, including human serum, porcine spermatozoa, human saliva, and urinary extracellular vesicles, detecting 33, 39, 49, and 37 O-glycans, respectively, thereby demonstrating its broad applicability.

Single cell glycan-linkages profiling for hepatocellular carcinoma early diagnosis using lanthanide encoded bacteriophage MS2 based ICP-MS.

Early diagnosis is paramount for enhancing survival rates and prognosis in the context of malignant diseases. Hepatocellular carcinoma (HCC), the second leading cause of cancer-related deaths worldwide, poses significant challenges for its early detection. In this study, we present an innovative approach which contributed to the early diagnosis of HCC. By lanthanide encoding signal amplification to map glycan-linkages at the single-cell level, the minute quantities of "soft" glycan-linkages on single cell surface were converted into "hard" elemental tags through the use of an MS2 signal amplifier. Harnessing the power of lanthanides encoded within MS2, we achieve nearly three orders of magnitude signal amplification. These encoded tags are subsequently quantified using single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS). Linear discriminant analysis (LDA) identifies seven specific glycan-linkages (α-2,3-Sia, α-Gal, α-1,2-Fuc, α-1,6-Fuc, α-2,6-Sia, α-GalNAc, and Gal-ß-1,3-GalNAc) as biomarkers. Our methodology is initially validated at the cellular level with 100% accuracy in discriminating between hepatic carcinoma HepG2 cells and their normal HL7702 cells. We apply this approach to quantify and classify glycan-linkages on the surfaces of 55 clinical surgical HCC specimens. Leveraging these seven glycan-linkages as biomarkers, we achieve precise differentiation between 8 normal hepatic specimens, 40 early HCC specimens, and 7 colorectal metastasis HCC specimens. This pioneering work represents the first instance of employing single-cell glycan-linkages as biomarkers promising for the early diagnosis of HCC with a remarkable 100% predictive accuracy rate, which holds immense potential for enhancing the feasibility and precision of HCC diagnosis in clinical practice.

Comparative analysis of Herceptin N-Linked glycosylation by HILIC-FLD and LC-MS/MS methods.

Monoclonal antibodies like Herceptin play a pivotal role in modern therapeutics, with their glycosylation patterns significantly influencing their bioactivity. To characterize the N-glycan profile and their relative abundance in Herceptin, we employed two analytical methods: hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) for released glycans and liquid chromatography tandem mass spectrometry (LC-MS/MS) for glycopeptides. Our analysis included 21 European Union (EU)-Herceptin lots and 14 United States (US)-Herceptin lots. HILIC-FLD detected 25 glycan species, including positional isomers, revealing comparable chromatographic profiles for both EU and US lots. On the other hand, LC-MS/MS identified 26 glycoforms within the glycopeptide EEQYNSTYR. Both methods showed that a subset of glycans dominated the total abundance. Notably, EU-Herceptin lots with an expiration date of October 2022 exhibited increased levels of afucosylated and high mannose N-glycans. Our statistical comparisons showed that the difference in quantitative results between HILIC-FLD and LC-MS/MS is significant, indicating that the absolute quantitative values depend on the choice of the analytical method. However, despite these differences, both methods demonstrated a strong correlation in relative glycan proportions. This study contributes to the comprehensive analysis of Herceptin's glycosylation, offering insights into the influence of analytical methods on glycan quantification and providing valuable information for the biopharmaceutical industry.

Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.

RATIONALE: The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI. METHODS AND RESULTS: To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 µm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively. CONCLUSIONS: Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.

Extraction, structure and antioxidant activity of the polysaccharides from morels (Morchella spp.): A review.

Morels (Morchella spp.), which are cultivated only in a few regions of the world, are edible mushrooms known for their various properties including antioxidation, immune regulation, antiinflammation, and antitumor effects. Polysaccharides from Morchella are principally responsible for its antioxidant activity. This paper reviews the extraction, purification, structural analysis and antioxidant activity of Morchella polysaccharides (MPs), providing updated research progress. Meanwhile, the structural-property relationships of MPs were further discussed. In addition, based on in vitro and in vivo studies, the major factors responsible for the antioxidant activity of MPs were summarized including scavenging free radicals, reduction capacity, inhibitory lipid peroxidation activity, regulating the signal transduction pathway, reducing the production of ROS and NO, etc. Finally, we hope that our research can provide a reference for further research and development of MPs.

Profiling of aberrant sialylated N-glycans in hepatocellular carcinoma by liquid chromatography mass spectrometry.

BACKGROUND: Aberrant sialylation is closely associated with the tumorigenesis, progression, and metastasis, and may be of importance for disease diagnosis. However, the analysis of altered expression of sialylated glycans (SGs) in blood is particularly challenging due to the low content and poor ionization efficiency of sialylated glycans in mass spectrometry. METHODS: An analytical strategy based on enrichment of SGs, liquid chromatography-high resolution mass spectrometric detection, and automatic glycan annotation was developed to profile the sialylated N-glycome in serum. The enrichment of sialylated glycans was accomplished using cationic cotton via electrostatic and hydrogen interaction. Using partial least squares-discriminant analysis (PLS-DA), the approach was applied for nontarget screening and profiling of aberrant sialylated N-glycans in hepatocellular carcinoma (HCC). RESULTS: 55 SGs were identified in human serum, and three important SGs (SG35, SG45, and SG46) were screened to have good diagnostic specificity for HCC. Their areas under the receiver operating characteristic (ROC) curve (AUC) were higher than α-fetoprotein (AFP)'s (AUC = 0.85), at 0.88, 0.87, and 0.91, respectively. When three SGs are combined, the diagnostic specificity for HCC may increase to 94 %. The fact that SGs biomarkers are sensitive to AFP-Negative HCC is very noteworthy. CONCLUSIONS: The method significantly advanced the search for sialylated glycan-based cancer biomarkers. In comparison to traditional indicators like AFP and imaging tools, SGs showed a higher diagnostic sensitivity for HCC.

Negative Charge-Carrying Glycans Attached to Exosomes as Novel Liquid Biopsy Marker.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more nega tive zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

Characterization of Concanavalin A-based lectin sorbents for the extraction of the human chorionic gonadotropin glycoforms prior to analysis by nano liquid chromatography-high resolution mass spectrometry.

Human chorionic gonadotropin (hCG) is constituted of the hCGα and hCGß subunits and is a highly glycosylated protein. Affinity supports based on immobilized Concanavalin A (Con A) lectin were used in solid phase extraction (SPE) to fractionate the hCG glycoforms according to their glycosylation state. For the first time, the lectin SPE fractions were off-line analysed by a nano liquid chromatography - high-resolution mass spectrometry (nanoLC-HRMS) method keeping the glycoforms intact. For this, home-made Con A sorbents were prepared by immobilizing lectin on Sepharose with a mean grafting yield of 98.2% (relative standard deviation (RSD) of 3.5%, n = 15). A capacity of about 100 µg of purified urinary hCG (uhCG) per ml of sorbent, grafted with a density of 10 mg of Con A per ml, was estimated. Average extraction yields of around 60% for both hCGα and hCGß glycoforms were obtained after optimization of the extraction protocol. Intra- and inter-assay evaluation led to average RSD values of around 10%, indicating a repeatable extraction procedure. Similar results were obtained with commercial Con A-based sorbents but only after their 3rd use or after an extensive pre-conditioning step. Finally, the Con A SPE led to the fractionation of some glycoforms of uhCG, allowing the detection of an hCGα glycoform with two tetra-antennary N-glycans that couldn't be detected by direct analysis in nanoLC-HRMS without Con A SPE. Regarding a recombinant hCG, a fractionation was also observed leading to the detection of unretained hCGα glycoforms with tri-antennary N-glycans. Therefore, the combination of lectin SPE with intact protein analysis by nanoLC-HRMS can contribute to a more detailed glycosylation characterization of the hCG protein.

Can We Boost N-Glycopeptide Identification Confidence? Smart Collision Energy Choice Taking into Account Structure and Search Engine.

High confidence and reproducibility are still challenges in bottom-up mass spectrometric N-glycopeptide identification. The collision energy used in the MS/MS measurements and the database search engine used to identify the species are perhaps the two most decisive factors. We investigated how the structural features of N-glycopeptides and the choice of the search engine influence the optimal collision energy, delivering the highest identification confidence. We carried out LC-MS/MS measurements using a series of collision energies on a large set of N-glycopeptides with both the glycan and peptide part varied and studied the behavior of Byonic, pGlyco, and GlycoQuest scores. We found that search engines show a range of behavior between peptide-centric and glycan-centric, which manifests itself already in the dependence of optimal collision energy on m/z. Using classical statistical and machine learning methods, we revealed that peptide hydrophobicity, glycan and peptide masses, and the number of mobile protons also have significant and search-engine-dependent influence, as opposed to a series of other parameters we probed. We envisioned an MS/MS workflow making a smart collision energy choice based on online available features such as the hydrophobicity (described by retention time) and glycan mass (potentially available from a scout MS/MS). Our assessment suggests that this workflow can lead to a significant gain (up to 100%) in the identification confidence, particularly for low-scoring hits close to the filtering limit, which has the potential to enhance reproducibility of N-glycopeptide analyses. Data are available via MassIVE (MSV000093110).

Extraction, purification, structural characterization and pharmacological activities of polysaccharides from sea buckthorn (Hippophae rhamnoides L.): A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Sea buckthorn (Hippophae rhamnoides L.) is an edible fruit with a long history in China as a medicinal plant. The fruits of H. rhamnoides are rich in a variety of nutrients and pharmacological active compounds. As one of the most important active ingredients in sea buckthorn, polysaccharides have attracted the attention of researchers due to their antioxidant, anti-fatigue, and liver protective qualities. AIM OF THE REVIEW: This review summarizes recent studies on extraction, purification, structural characterization and pharmacological activities of polysaccharides from sea buckthorn. In addition, the relationship between the structure and the activities of sea buckthorn polysaccharides (SBPS) were discussed. This review would provide important research bases and up-to-date information for the future in-depth development and application of sea buckthorn polysaccharides in the field of pharmaceuticals and functional foods. MATERIALS AND METHODS: By inputting the search term "Sea buckthorn polysaccharides", relevant research information was obtained from databases such as Web of Science, Google Scholar, PubMed, China Knowledge Network (CNKI), China Master Theses Full-text Database, and China Doctoral Dissertations Full-text Database. RESULTS: The main extraction methods of SBPS include hot water extraction (HWE), ultrasonic assisted extraction (UAE), microwave-assisted extraction (MAE), flash extraction (FE), and ethanol extraction. More than 20 polysaccharides have been isolated from sea buckthorn fruits. The chemical structures of sea buckthorn polysaccharides obtained by different extraction, isolation, and purification methods are diverse. Polysaccharides from sea buckthorn display a variety of pharmacological properties, including antioxidant, anti-fatigue, liver protection, anti-obesity, regulation of intestinal flora, immunoregulation, anti-tumor, anti-inflammatory, and hypoglycemic activities. CONCLUSIONS: Sea buckthorn has a long medicinal history and characteristics of an ethnic medicine and food. Polysaccharides are one of the main active components of sea buckthorn, and they have received increasing attention from researchers. Sea buckthorn polysaccharides have remarkable pharmacological activities, health benefits, and broad application prospects. In addition, further exploration of the chemical structure of SBPS, in-depth study of their pharmacological activities, identification of their material basis, characterization of disease resistance mechanisms, and potential health functions are still directions of future research. With the accumulation of research on the extraction and purification processes, chemical structure, pharmacological effects, molecular mechanisms, and structure-activity relationships, sea buckthorn polysaccharides derived from natural resources will ultimately make significant contributions to human health.

Noninvasive serum N-glycans associated with ovarian cancer diagnosis and precancerous lesion prediction.

BACKGROUND: Ovarian cancer (OC) is one of the most common gynecological tumors with high morbidity and mortality. Altered serum N-glycome has been observed in many diseases, while the association between serum protein N-glycosylation and OC progression remains unclear, particularly for the onset of carcinogenesis from benign neoplasms to cancer. METHODS: Herein, a mass spectrometry based high-throughput technique was applied to characterize serum N-glycome profile in individuals with healthy controls, benign neoplasms and different stages of OC. To elucidate the alterations of glycan features in OC progression, an orthogonal strategy with lectin-based ELISA was performed. RESULTS: It was observed that the initiation and development of OC was associated with increased high-mannosylationand agalactosylation, concurrently with decreased total sialylation of serum, each of which gained at least moderately accurate merits. The most important individual N-glycans in each glycan group was H7N2, H3N5 and H5N4S2F1, respectively. Notably, serum N-glycome could be used to accurately discriminate OC patients from benign cohorts, with a comparable or even higher diagnostic score compared to CA125 and HE4. Furthermore, bioinformatics analysis based discriminative model verified the diagnostic performance of serum N-glycome for OC in two independent sets. CONCLUSIONS: These findings demonstrated the great potential of serum N-glycome for OC diagnosis and precancerous lesion prediction, paving a new way for OC screening and monitoring.

Sulfated polysaccharides of Laetiporus sulphureus fruiting bodies exhibit anti-breast cancer activity through cell cycle arrest, apoptosis induction, and inhibiting cell migration.

ETHNOPHARMACOLOGICAL RELEVANCE: Laetiporus sulphureus has long been used as an edible and medicinal mushroom in Asia, America, and Europe. Its fruiting bodies are widely used in folk medicine for treating cancer, gastric diseases, cough, and rheumatism. Polysaccharides are an important bioactive component of mushrooms. In nature, sulfated polysaccharides have never been reported in mushrooms. Furthermore, there is no information on differences in physicochemical properties and anti-breast cancer activities between polysaccharides (PS) and sulfated polysaccharides (SPS) of L. sulphureus. AIM OF THE STUDY: This study aimed to investigate the physicochemical properties of PS and SPS isolated from fruiting bodies of L. sulphureus and examine their anti-proliferative effects and mechanism(s) of action on MDA-MB-231 breast cancer cells. METHODS: Polysaccharides (PS) were isolated using hot water and ethanol precipitation methods. Sulfated polysaccharides (SPS) were isolated by the papain-assisted hydrolysis method. Physicochemical properties comprising sugar, protein, uronic acid, and sulfate contents, and molecular weight, monosaccharide composition, and structural conformation were analyzed on PS and SPS. In the anti-cancer study, a triple-negative breast cancer cell line (MDA-MB-231) and a normal human mammary epithelial cell line (H184B5F5/M10) were used to evaluate the anti-proliferative activity of PS and SPS, and their mechanism(s) of action. RESULTS: The results showed that SPS, which had higher sulfate and protein contents and diversified monosaccharide composition, exhibited more potent anti-proliferative activity against MDA-MB-231 cells than PS. Furthermore, it had a selective cytotoxic effect on breast cancer cells but not the normal cells. SPS induced cell cycle arrest at G0/G1 phase via down-regulating CDK4 and cyclin D1 and up-regulating p21 protein expression. Breast cancer cell apoptosis was not observed until 72 h after SPS treatment. In addition, SPS also markedly inhibited breast cancer cell migration. CONCLUSION: This study demonstrates that SPS exhibited selective cytotoxicity and was more potent than PS in inhibiting MDA-MB-231 cell proliferation. The contents of sulfate and protein, and monosaccharide composition could be the main factors affecting the anti-breast cancer activity of L. sulphureus SPS.

Exploring apple pectic polysaccharides: Extraction, characterization, and biological activities - A comprehensive review.

Apple (Malus domestica) is a popular and ancient fruit of the Myrtaceae family. Apple fruit is well-known for its great nutritional and phytochemical content consisted of beneficial compounds such as polyphenols, polysaccharides, sterols, and organic acids. Polysaccharides extracted from different parts of the apple fruit, including the peel, pomace, or the whole fruit, have been extensively studied. Researchers have investigated the structural characteristics of these polysaccharides, such as molecular weight, type of monosaccharide unit, type of linkage and its position and arrangement. Besides this, functional properties and physicochemical and of apple polysaccharides have also been studied, along with the effects of extraction procedures, storage, and processing on cell wall polysaccharides. Various extraction techniques, including hot water extraction, enzymatic extraction, and solvent-assisted extraction, have been studied. From the findings, it was evident that apple polysaccharides are mainly composed of (1 â†’ 3), (1 â†’ 6): α-ß-glycosidic linkage. Moreover, the apple polysaccharides were demonstrated to exhibit antioxidant, hepatoprotective, anti-cancer, hypoilipidemic, and enzyme inhibitory properties in vitro and in vivo. The potential applications of apple polysaccharides in the food, cosmetic, pharmaceutical, nutraceutical industries have also been explored in the present review. Overall, the research on apple polysaccharides highlights their significant potential as a source of biologically active compounds with various health benefits and practical applications.

Highly sensitive quantitative detection of glycans on exosomes in renal disease serums using fluorescence signal amplification strategies.

Exosomal glycoproteins play a significant role in many physiological and pathological processes. However, the detection of exosome surface glycans is currently challenged by the complexity of biological samples or the sensitivity of the methods. Herein, we prepared a novel fluorescent probe of biotin-functionalized nanocrystals (denoted as CdTe@cys-biotin) and applied it for the first time for the detection of the expression of exosomal surface glycans using a fluorescence amplification strategy. First, the dual affinity of TiO2 and CD63 aptamers of Fe3O4@TiO2-CD63 was utilized to rapidly and efficiently capture exosomes within 25 min. In this design, interference from other vesicles and soluble impurities can be avoided due to the dual recognition strategy. The chemical oxidation of NaIO4 oxidized the hydroxyl sites of exosomal surface glycans to aldehydes, which were then labeled with aniline-catalyzed biotin hydrazide. Using the high affinity between streptavidin and biotin, streptavidin-FITC and probes were successively anchored to the glycans on the exosomes. The fluorescent probe achieved the dual function of specific recognition and fluorescent labeling by modifying biotin on the surface of nanocrystals. This method showed excellent specificity and sensitivity for exosomes at concentrations ranging from 3.30 × 102 to 3.30 × 106 particles/mL, with a detection limit of 121.48 particles/mL. The fluorescent probe not only quantified exosomal surface glycans but also distinguished with high accuracy between serum exosomes from normal individuals and patients with kidney disease. In general, this method provides a powerful platform for sensitive detection of exosomes in cancer diagnosis.

High-throughput analysis of glycan sorting into extracellular vesicles.

Extracellular vesicles (EVs) are cell-released vesicles that mediate intercellular communication by transferring bioactive cargo. Protein and RNA sorting into EVs has been extensively assessed, while selective enrichment of glycans in EVs remains less explored. In this study, a mass spectrometry-based approach, glycan node analysis (GNA), was applied to broadly assess the sorting of glycan features into EVs. Two metastatic variants (lung and bone) generated in mouse modes from the MDA-MB-231 human breast cancer cell line were assessed, as these EVs are known to contain distinct organotropic biomolecules. EVs were isolated from conditioned cell culture medium by tangential flow filtration and authenticated by standard techniques. GNA analysis revealed selective enrichment of several glycan features in EVs compared to the originating cells, particularly those associated with binding to the extracellular matrix, which was also observed in EVs from the parental MDA-MB-231 cell line (human pleural metastases). The bone-tropic variant displayed enrichment of distinct EV glycan features compared to the lung-tropic one. Additionally, the metastatic variants generated in mouse models displayed reduced EV glycan sorting compared to the parental metastatic cell line. This study represents the first comprehensive assessment of differences in glycan features between EVs and originating cells and provides evidence that the diversity of EV glycan sorting is reduced upon generation of variant cell lines in mouse models. Future research is likely to uncover novel mechanisms of EV glycan sorting, shed light on glycan features for EV authentication or biomarker purposes, and assess functional roles of the EV glycocode in (patho)physiology.

Serum-based Comprehensive N-Glycans Profiling Analysis in Different Gastric Disease Stages by Porous Graphitic Carbon Liquid Chromatography-Mass Spectrometry Associated With Potential Marker Discovery.

BACKGROUND/AIM: N-glycans are potential serum biomarkers due to their aberrant structure and abundance alteration during disease progression. Few studies have been associated with relative quantitative N-glycans profiling during different gastric disease stages. In this study, we conducted an investigation on the profiling of N-glycans in patients with gastric disease, as well as in healthy controls. MATERIALS AND METHODS: In this study, the porous graphitization carbon chromatography-high resolution Fourier transform mass spectrometry (PGC-FTMS) method was applied to assess comprehensive N-glycans profiling in patients at different stages of gastric disease, including gastritis, atrophic gastritis, gastric ulcer, gastric polyps, and gastric cancer. RESULTS: A total of 45 N-glycans (relative abundance >0.1%) were detected, and 9 N-glycans were found to be potential biomarkers for gastric disease detection. Along with the progression of gastric disease, the abundance of sialylated N-glycans increased, while that of core-fucosylated N-glycans decreased. Multivariate statistical analysis demonstrated that N-glycans profiling between gastritis and healthy controls had significant differences. The characteristic N-glycans distinguished gastric cancer from healthy controls, which had strong clinical diagnostic value. CONCLUSION: The relative quantitative profile of N-glycans in different gastric disease stages was revealed and serum N-glycans are proposed for distinguishing gastric disease stages in clinical application.

Structural Analysis and Characterization of an Antiproliferative Lectin from Canavalia villosa Seeds.

Cells use glycans to encode information that modulates processes ranging from cell-cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin's specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways.

Imaging of N-Linked Glycans in Biological Tissue Sections Using Nanospray Desorption Electrospray Ionization (nano-DESI) Mass Spectrometry.

N-linked glycans are complex biomolecules vital to cellular functions that have been linked to a wide range of pathological conditions. Mass spectrometry imaging (MSI) has been used to study the localization of N-linked glycans in cells and tissues. However, their structural diversity presents a challenge for MSI techniques, which stimulates the development of new approaches. In this study, we demonstrate for the first time spatial mapping of N-linked glycans in biological tissues using nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI). Nano-DESI MSI is an ambient ionization technique that has been previously used for imaging of metabolites, lipids, and proteins in biological tissue samples without special sample pretreatment. N-linked glycans are released from glycoproteins using an established enzymatic digestion with peptide N-glycosidase F, and their spatial localization is examined using nano-DESI MSI. We demonstrate imaging of N-linked glycans in formalin-fixed paraffin-embedded human hepatocellular carcinoma and human prostate tissues in both positive and negative ionization modes. We examine the localization of 38 N-linked glycans consisting of high mannose, hybrid fucosylated, and sialyated glycans. We demonstrate that negative mode nano-DESI MSI is well-suited for imaging of underivatized sialylated N-linked glycans. On-tissue MS/MS of different adducts of N-linked glycans proves advantageous for elucidation of the glycan sequence. This study demonstrates the applicability of liquid extraction techniques for spatial mapping of N-linked glycans in biological samples, providing an additional tool for glycobiology research.

Evaluation of antibody-based single cell type imaging techniques coupled to multiplexed imaging of N-glycans and collagen peptides by matrix-assisted laser desorption/ionization mass spectrometry imaging.

The integration of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with single cell spatial omics methods allows for a comprehensive investigation of single cell spatial information and matrisomal N-glycan and extracellular matrix protein imaging. Here, the performance of the antibody-directed single cell workflows coupled with MALDI-MSI are evaluated. Miralys™ photocleavable mass-tagged antibody probes (MALDI-IHC, AmberGen, Inc.), GeoMx DSP® (NanoString, Inc.), and Imaging Mass Cytometry (IMC, Standard BioTools Inc.) were used in series with MALDI-MSI of N-glycans and extracellular matrix peptides on formalin-fixed paraffin-embedded tissues. Single cell omics protocols were performed before and after MALDI-MSI. The data suggests that for each modality combination, there is an optimal order for performing both techniques on the same tissue section. An overall conclusion is that MALDI-MSI studies may be completed on the same tissue section as used for antibody-directed single cell modalities. This work increases access to combined cellular and extracellular information within the tissue microenvironment to enhance research on the pathological origins of disease.