Background:Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) are progressive neurodegenerative diseases (NDs) characterized by chronic neuronal loss. The lack of effective treatments highlights the urgent need for reliable lipid biomarkers to enable diagnosis and monitor disease progression. Previous lipidomic investigations of altered lipid metabolism have focused on a single disease type, limiting cross-disease comparisons. Methods:We applied the untargeted liquid chromatography-mass spectrometry (LC/MS) technique to profile brain lipidome alterations and to identify disease-specific lipid biomarkers across AD, HD, and PD. Brain tissue samples were collected from four cerebral lobes of healthy volunteers (HV, n = 24) and patients diagnosed with AD (n = 24), PD (n = 24), and HD (n = 24). All groups include three males and three females, with brain tissues from four cortical regions sacrificed from each individual. Results:A total of 243 lipid molecular species spanning five major classes were annotated, revealing distinct disease-specific lipidomic profiles that differentiated HV from the AD, HD, and PD groups via multivariate analysis. Sphingomyelins and oxidized phosphatidylserine [PS (16:1/24:0;O1)] were significantly increased, while lysophosphatidylcholines (LPC 18:2, LPC 17:2) were decreased in the AD group relative to HV. HD exhibited elevated PS (O-17:0/22:6) and ω-6 fatty acid esterified cholesteryl esters (CE 18:2, CE 20:4), alongside decreased essential neuronal lipids such as phosphatidylinositols (PI). The PD lipidome alterations closely resembled those of HD, indicating partially overlapping disruptions in brain lipid metabolism. Receiver operating characteristic analysis identified PS (16:1/24:0;O1), PS (O-17:0/22:6), and PI (18:1/18:1) as potential discriminatory biomarkers with strong diagnostic performance. Regional heatmap analysis revealed significant lipid perturbations were observed in the parietal and occipital lobes across all NDs. Conclusion:This study provides a comprehensive overview of disease- and region-specific alterations in the brain lipidome of AD, HD, and PD. The identified lipid species-PS (16:1/24:0;O1), PS (O-17:0/22:6), and PI (18:1/18:1)-may serve as promising candidate biomarkers for NDs diagnosis and warrant further mechanistic and longitudinal validation with large data set.

Yersinia pseudotuberculosis is a zoonotic gram-negative bacterium with a wide range of hosts. Twenty-one classifications of O-antigens of Y. pseudotuberculosis, including their glyco-subtypes, have been reported and used as epidemiological indicators of health damage caused by this bacterium. In this study, we performed rapid identification of Y. pseudotuberculosis O-antigens using a modified MALDI O-antigen glycotyping method. By employing a DAN/DHB/K matrix, the O-antigen repeating units were detected as potassium adduct ions, facilitating the discrimination between hexoses, deoxyhexoses, and dideoxyhexoses, compared to the conventional matrix system with sodium ions. The branched dideoxyhexose of Y. pseudotuberculosis O-antigens was eliminated by the pretreatment or ionization process of MALDI glycotyping, and the repeating unit pattern reflecting the main-chain glycan sequence was given as the main spectrum. Of the four strains investigated, O2b and O3 polysaccharides with the same main-chain glycan sequence gave characteristic repeating unit pattern peaks, reflecting the difference in the position and stereochemistry of the branching dideoxyhexose. This study will accelerate the implementation of intraspecies classification targeting O-antigens in microbial identification techniques using MALDI-TOF MS.

Understanding characteristic post-source decay (PSD) fragmentation patterns in tandem mass spectrometry (MS/MS) is important for the identification of target molecules. In this study, we explored the characteristic PSD patterns associated with O-linked glycopeptides and their cyclization using the MALDI-TOF/TOF MS analysis of linear and cyclic antifreeze glycoproteins. We performed a comparative analysis of the proton and sodium adduct ions of the peptide backbones of antifreeze glycoproteins, which have a simple repeating sequence, shedding light on the characteristics of the fragmentation of the threonine side chain and that of its cyclized form. Furthermore, the presence or absence of a glycan on the threonine side chain and its substitution with serine caused changes in its fragmentation. These findings are expected to contribute to the prediction of three-dimensional peptide structures and the search for physiologically active O-linked glycopeptides and cyclic (glyco)peptides.

O-linked glycopeptides have attracted attention because of their ability to control peptide conformation and function via glycosylation. Cyclic peptides have also attracted attention for their various physiological and physical activities, owing to the conformational constraints associated with cyclization. We found that cyclic antifreeze glycopeptides showed characteristic peptide chain fragmentation patterns in the MALDI-TOF/TOF MS analysis. The fragmentation patterns were characterized by the parallel occurrence of side-chain fragmentation of threonine residues and fragmentation within the macrocyclic peptide bond associated with ring opening. Furthermore, the tendency for side chain fragmentation was altered depending on the adduct ions, the presence or absence of glycans, and the substitution of threonine with serine. These fragmentation mechanisms have been suggested to be influenced by conformational changes in cyclic peptides, along with adduct ion coordination or structural modifications, particularly threonine residues. This finding is expected to contribute to the prediction of peptide conformation based on fragmentation tendencies and to the search for physiologically active O-linked glycopeptides and cyclic (glyco)peptides.

Breast cancer (BC) is a major global health concern, and early detection is key to improving patient outcomes. Aberrant glycosylation, particularly the sulfation of glycans, is implicated in cancer progression; however, analyzing these low-abundance glycans is challenging. This study aimed to profile serum sulfated N-glycans in Ethiopian patients with BC to identify novel biomarkers for the early detection of BC. Using a glycoblotting-based sulphoglycomics workflow, including high-throughput glycoblotting enrichment, weak anion exchange (WAX) separation, and MALDI-TOF MS, serum samples from 76 BC patients and 20 healthy controls were analyzed. Statistical evaluation revealed significant differences in the sulfated N-glycan profiles. Seven mono-sulfated N-glycans were markedly elevated in patients with BC, demonstrating high diagnostic accuracy (AUC ≥ 0.8) in this internal cohort. Terminal Lewis-type glycan epitopes were prominent in sulfated glycans but were absent in their non-sulfated counterparts. The increased fucosylation and sialylation of sulfated glycans are statistically significant markers of early-stage BC. The preservation of sialic acid groups during the analysis ensured detailed structural insight. This pioneering study quantitatively examined sulfated N-glycans in BC and identified potential glyco-biomarkers for early detection. Validation in larger, diverse cohorts is needed to establish their broader diagnostic relevance and improve our understanding of cancer-associated glycomic alterations.

Alterations in sulfated glycans are associated with several pathological conditions, including cancer. However, analysis of sulfated glycans poses challenges, making the investigation of sulfated glycan profiles a topic of significant interest in the search for novel biomarkers for early BC detection. We used a glycoblotting-based sulphoglycomics workflow to examine sulfated N-glycans present in the serum of Ethiopian patients with BC. Seven mono-sulfated glycans were significantly upregulated in the sera of BC patients compared to the control group. Each identified glycan showed significant abundance with AUC ≥ 0.8 and demonstrated high diagnostic accuracy in predicting early stage BC patients. Importantly, the sulfated glycans were analyzed without removing the sialic group, allowing for comprehensive evaluation of the sialylation status of the identified sulfated glycans. This study represents the first quantitative analysis of sulfated N-glycans in patients with BC and identifies novel biomarkers with discriminatory potential in the early stages of BC. Statement of significance This study presents a quantitative analysis of sulfated N-glycans in BC, aiming to identify novel glyco-biomarkers that demonstrate high diagnostic accuracy for early stage BC. Analyzing sulfated glycans without removing sialic acids offers comprehensive insights. These findings advance the understanding of BC, potentially enhance early detection, and improve patient outcomes. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement S. Tebeje reports grant from Addis Ababa University for Breast Cancer Thematic Research. H. Hinou reports grants from JSPS KAKENHI and Core-to-Core B. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Informed consent was obtained from all study participants and the study was conducted in accordance with the ethical standards outlined in the Declaration of Helsinki. The ethical review boards of Addis Ababa University, School of Medicine, Ethiopia, and Hokkaido University, Faculty of Advanced Life Sciences, Japan approved the research protocols. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data are available in the main text or supplementary materials.

Sulfated N-glycans play a crucial role in the interaction between influenza A virus (IAV) and its host. These glycans have been found to enhance viral replication, highlighting their significance in IAV propagation. This study investigated the expression of acidic N-glycans, specifically sulfated and phosphorylated glycans, in the egg whites of 72 avian species belonging to the Order Anseriformes (waterfowls). We used the glycoblotting-based sulphoglycomics approach to elucidate the diversity of acidic N-glycans and infer their potential role in protecting embryos from infections. Family-specific variations in sulfated and phosphorylated N-glycan profiles were identified in waterfowl egg whites. Different waterfowl species exhibited distinct expressions of sulfated trans-Gal(+) and trans-Gal(-) N-glycan structures. Additionally, species-specific expression of phosphorylated N-glycans was observed. Furthermore, it was found that waterfowl species with high avian influenza virus (AIV) prevalence displayed a higher abundance of phosphorylated hybrid and high-mannose N-glycans on their egg whites. These findings shed light on the importance of phosphorylated and sulfated N-glycans in understanding the role of acidic glycans in IAV propagation.

Escherichia coli O111 is a critical pathogenic E. coli serotype that causes severe, potentially fatal complications. Despite its reported variation, only one structure of the O-antigen polysaccharide from E. coli O111 has been reported. Here, a substructure of the O-antigen from E. coli O111 was characterized using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and NMR analysis. MALDI glycotyping revealed differing O-antigen repeating unit masses of Δm/z 787 and 828 in the E. coli strains and lipopolysaccharides from the O111 serogroup. This variation was caused by the replacement of the hexose residue with hexosamine in the repeating units, which was further confirmed by LIFT-TOF/TOF analysis. Structural elucidation of the O111 substructure by NMR analysis further demonstrated replacement of the hydroxyl group with an N-acetyl group on the terminal glucose residue of the O-antigen pentasaccharide repeating unit. To our knowledge, this study is the first to provide a detailed structural analysis of a new O-antigen substructure from the E. coli O111 serogroup.

Abstract Background Breast cancer (BC) is a significant global health concern among women, and early detection plays a pivotal role in enhancing patient survival rates. Alterations in the structure and abundance of sulfated glycans have been linked to various diseases including cancer. However, due to their low abundance, analyzing sulfated glycans poses challenges, making the investigation of sulfated glycan profiles a topic of significant interest in the search for novel biomarkers for early BC detection. Methods In this study, we utilized a glycoblotting-based sulphoglycomic workflow to examine the presence of sulfated N-glycans in the serum of Ethiopian patients with BC. This approach integrates high-throughput glycoblotting enrichment technology, WAX separation, and MALDI-TOF MS. The sulfated N-glycan profiles in the whole serum of 76 BC patients and 20 age-matched healthy controls were analyzed. Results The findings revealed that seven monosulfated glycans were significantly upregulated in the serum of BC patients compared to that in the control group. Each identified glycan showed significant abundance with an AUC ≥ 0.8 and demonstrated high diagnostic accuracy in predicting early-stage BC patients. Sulfated glycans abundantly displayed terminal Lewis-type glycan epitopes, unlike their negligible presence in nonsulfated N-glycans in serum, whose abundance has been strongly associated with BC progression, metastasis, and immune invasion. Importantly, sulfated glycans were analyzed without removing the sialic group, allowing for a comprehensive evaluation of the sialylation status of the identified sulfated glycans. Conclusion To the best of our knowledge, this study represents the first quantitative analysis of sulfated N-glycans in patients with BC, identifying novel glyco-biomarkers with discriminatory potential in the early stages of BC.

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis ‐binding and trans ‐bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)‐1, −3, −4, and −9 variant test panels, achieved via rational protein engineering, and a synthetic α‐dystroglycan (DG) O ‐Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design‐functionality relationships within this lectin family. Enhancement of prototype Gal‐1 and chimera‐type Gal‐3 cis ‐binding toward the prepared ligands is possible by transforming these lectins into tandem‐repeat type and prototypes, respectively. Furthermore, Gal‐1 variants demonstrated improved trans ‐bridging capabilities between core M1 α‐DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of α‐dystroglycanopathy.

Sulfated N- and O-glycans exist in trace levels which are challenging to detect, especially when abundant neutral and sialylated glycans are present. Current matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based sulfoglycomics approaches effectively utilize permethylation to discriminate sulfated glycans from sialyl-glycans. And a charge-based separation to isolate the sulfated glycans from the rest of the permethylated neutral and sialyl-glycans. However, these approaches suffer from concomitant sample losses during cleanup steps. Herein, we describe Glycoblotting as a straightforward complementary method with seamless glycan purification, enrichment, methylation, and labeling on a single platform to address sulfated glycan enrichment, sialic acid methylation, and sample loss. Glycoblottings' on-bead chemoselective ligation of reducing sugars with hydrazide showed excellent recovery of sulfated glycans, allowing the detection of more sulfated glycan species. On-bead methyl esterification of sialic acid using 3-methyl-1-p-tolyltriazene (MTT) effectively discriminates sulfated glycans from sialyl-glycans. Furthermore, we have shown that using MTT as a methylating agent allowed us to simultaneously detect and differentiate sulfate from phosphate groups in isobaric N-glycan species. We believe that Glycoblotting will contribute significantly to the MALDI-TOF MS-based Sulphoglycomics workflow.

3-Amino-4-hydroxybenzoic acid (AHB) was the first matrix identified by glycoprotein glycan analysis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, compared to commonly used matrices, such as 2,5-dihydroxybenzoic acid (DHB), AHB is less efficient at glycan ionization and lacks the ability to ionize other molecular species, such as peptides, and thus is no longer used. In this study, we focused on the glycan-selective ionization ability of AHB and its low-noise properties in the low-molecular-weight region, as we expected that these properties could be enhanced by adding sodium to AHB. Sodium-doped AHB (AHB/Na) selectively imparts sodium adduct ions onto O-glycan fragments generated by the in-source decay (ISD) of glycopeptides and glycoproteins containing O-glycans that occurs during intense laser irradiation, enabling direct O-glycan analysis. Furthermore, we demonstrated that it is possible to investigate the internal structure of each O-glycan fragment with pseudo-MS/MS/MS using the sodium adduct ion of the O-glycan-derived ISD fragments from an intact mucin mixture.

Seek a cell anchorage galectin bridge: Transforming the wildtype Galectin (Gal)-1 and -3 to a tandem-repeat type variant via rational protein engineering enhanced cis-binding activity with O-Mannosylated core M1 glycopeptides of α-dystroglycan (DG). Furthermore, the Gal-1 variants (but not Gal-3 variants) demonstrated trans-bridging activity with the prepared α-DG core-M1 glycoconjugates and laminins in situ via the carbohydrate-protein interactions. Thus, Gal-1 and its variants are exciting candidates for treating several forms of muscular dystrophy. More information can be found in the Research Article by H. Kaltner, H. Hinou, et al.

Matrix-assisted laser desorption/ionization (MALDI) is one of the soft ionization methods in mass spectrometry (MS). Its ana-lyte compatibility with a matrix can improve analyte-selective ionization efficiency and control the fragmentation properties of the product ions. 2,5-dihydroxybenzoic acid (DHB) is a versatile matrix that can be used for various molecular species. Although DHB is commonly used as the matrix of first choice for MALDI MS of glycans, many attempts to optimize DHB for glycan analysis using additives have also been reported. This mini-review will present additives of the DHB-based matrix that promote direct analysis of carbohydrates and glycoconjugates by MALDI MS.

Matrix-assisted laser desorption/ionization (MALDI) is one of the soft ionization methods in mass spectrometry (MS). Its analyte compatibility with a matrix can improve analyte-selective ionization efficiency and control the fragmentation properties of the product ions. 2,5-dihydroxybenzoic acid (DHB) is a versatile matrix that can be used for various molecular species. Although DHB is commonly used as the matrix of first choice for MALDI MS of glycans, many attempts to optimize DHB for glycan analysis using additives have also been reported. This mini-review will present additives of the DHB-based matrix that promote direct analysis of carbohydrates and glycoconjugates by MALDI MS.

Pancreatic cancer is highly metastatic and has poor prognosis, mainly due to delayed detection, often after metastasis has occurred. A novel method to enable early detection and disease intervention is strongly needed. Here we unveil for the first time that pancreatic cancer cells (PANC-1) and secreted exosomes express MUC1 bearing cancer-relevant dynamic epitopes recognized specifically by an anti-MUC1 antibody (SN-131), which binds specifically core 1 but not core 2 type O-glycans found in normal cells. Comprehensive assessment of the essential epitope for SN-131 indicates that PANC-1 cells produce dominantly MUC1 with aberrant O-glycoforms such as Tn, T, and sialyl T (ST) antigens. Importantly, SN-131 showed the highest affinity with MUC1 bearing ST antigen at the immunodominant DTR motif (K-D = 1.58 nM) independent of the glycosylation states of other Ser/Thr residues in the MUC1 tandem repeats. The X-ray structure revealed that SN-131 interacts directly with Neu5Ac and root GalNAc of the ST antigen in addition to the proximal peptide region. Our results demonstrate that targeting O-glycosylated "dynamic neoepitopes" found in the membrane-tethered MUC1 is a promising therapeutic strategy for improving the treatment outcome of patients with pancreatic cancer.

The rapid analysis of glycan patterns (glycoforms) of glycoproteins can accelerate their quality control and biomarker discovery. We have focused on the direct analysis of glycoprotein glycoforms using matrix-assisted laser desorption/ionization in-source decay mass spectrometry (MALDI-ISDMS), called MALDI glycotyping. Our results show that the 1,5-diaminonaphthalene (DAN)/2,5-dihydroxybenzoic acid (DHB)/Na matrix can directly analyze the glycoforms in the femtomolar range of intact glycoproteins. The addition of DAN improved the morphology of the solid matrix due to the mixture of DAN and DHB, which significantly contribute to the high sensitivity of this direct analysis. Adding DAN significantly improved the sensitivity of the glycan precursor ions in the TOF/TOF analysis because of its enhanced fragmentation effect as an efficient UV-MALDI matrix. Further, practical glycoform analysis (glycotyping) of diluted biological samples containing glycoproteins, such as egg whites, was also successfully achieved.

A simple matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) approach has been developed to analyze the structural pattern of glycan on glycoproteins without any digestive or modification pretreatment. For a 7pmol sample of intact glycoprotein, RNase B, a solid ionic matrix DHB-aniline-sodium enabled a regioselective in-source decay (ISD) and glycan-selective ionization as a sodium adduct ion, [M+Na](+). This matrix significantly reduced the formation of peptide-fragment-derived ions, and a high-mannose-type glycan pattern was observed. This ISD product ion was formed by selective cleavage of the reducing end of the glycan by (0,2)A or (2,4)A type cross-ring cleavage. Pseudo-MS3-type post-source decay (PSD) analysis of an A type ion indicated the regioselectivity of the ISD fragmentation and the glycan sequence. Combining ISD glycomics with conventional ISD proteomics will accelerate quality control for glycoprotein drugs and biomarker discovery.

Matrix selection is a critical factor for success in glycomics studies using matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). In this study, we evaluated and optimized a new solid ionic matrix—O-benzylhydroxylamine (BOA)/2,5-dihydroxybenzoic acid (DHB)/Na—containing BOA and a small amount of sodium as the counter salt of DHB. The concentration of a mixture of BOA/DHB/Na and glycans on a MALDI target plate led to O-benzyloxy tagging of the reducing ends of the glycans. The BOA/DHB/Na matrix showed excellent aggregation performance and the ability to form a homogeneous solid salt on the MALDI target plate with a water-repellent surface. In addition, the BOA/DHB/Na matrix showed a simple peak pattern with suppressed in-source and post-source decay of the reducing ends of the glycans, as well as improved ionization efficiency of glycans. Utilizing the characteristics of the BOA/DHB/Na matrix, O-glycan analysis of porcine stomach mucin showed excellent detection sensitivity and reproducibility of the peak patterns. This BOA/DHB/Na matrix can accelerate glycomics studies using MALDI–MS and, in combination with other organic salt-type matrices that we have developed, constitutes a valuable tool for glycomics studies.