Glycoproteomics: Charting new territory in mass spectrometry and glycobiology.

Glycosylation is an incredibly common and diverse post-translational modification that contributes widely to cellular health and disease. Mass spectrometry is the premier technique to study glycoproteins; however, glycoproteomics has lagged behind traditional proteomics due to the challenges associated with studying glycosylation. For instance, glycans dissociate by collision-based fragmentation, thus necessitating electron-based fragmentation for site-localization. The vast glycan heterogeneity leads to lower overall abundance of each glycopeptide, and often, ion suppression is observed. One of the biggest issues facing glycoproteomics is the lack of reliable software for analysis, which necessitates manual validation and serves as a massive bottleneck in data processing. Here, I will discuss each of these challenges and some ways in which the field is attempting to address them, along with perspectives on how I believe we should move forward.

Determination of Molecular Dimensions of Carbohydrate Polymers (Polysaccharides) by Size Exclusion Chromatography (SEC).

Calibrated size exclusion chromatography (SEC) is a useful tool for the analysis of molecular dimensions of polysaccharides. The calibration takes place with a set of narrow distributed dextran standards and peak position technique. Adapted columns systems and dissolving processes enable for the adequate separation of carbohydrate polymers. Plant-extracted fructan (a homopolymer with low molar mass and excellent water solubility) and mucilage (differently structured, high molar mass heteropolysaccarides that include existing supramolecular structures, and require a long dissolving time) are presented as examples of the versatility of this technique. Since narrow standards similar to the samples (chemically and structurally) are often unavailable, it must be noted that the obtained molar mass values and distributions by this method are only apparent (relative) values, expressed as dextran equivalents.

Atomic Force Microscopy in the Characterization of the Structure of Cell Wall Components.

Atomic force microscopy (AFM) has broken boundaries in the characterization of the supramolecular architecture of cell wall assemblies and single cell wall polysaccharides at the nanoscale level. Moreover, AFM provides an opportunity to evaluate the mechanical properties of cell wall material which is not possible with any other method. However, in the case of plant tissue, the critical step is a smart sample preparation that should not affect the polysaccharide structure or assembly and on the other hand should consider device limitations, especially scanner ranges. In this chapter, the protocols from the sample preparation, including isolation of cell wall material and extraction of cell wall polysaccharide fractions, through AFM imaging of polysaccharide assemblies and single molecules until an image analysis to obtain quantitative data characterizing the biopolymers are presented.

In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation.

The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.

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.

New insight from MALDI-TOF MS and multivariate data analysis on the botanical origin of polysaccharide-based paint binders in ancient Egypt.

Polysaccharide-based materials of plant origin are known to have been used as binding media in paint and ground layers of artifacts from ancient Egypt, including wall paintings, cartonnages and sarcophagi. The use of gums from Acacia, Astragalus and Prunus genera has been suggested in the literature on the basis of their qualitative or quantitative monosaccharide profile after complete chemical hydrolysis. The introduction of partial enzymatic digestion of the polysaccharide material, followed by analysis of the released oligosaccharides by matrix assisted laser desorption ionization-time-of-flight mass spectrometry, has proved effective in discriminating among gums from different genera, as well as among species within the Acacia genus. In this study, the previously built Acacia database was expanded, principal component analysis (PCA) was used to aid in grouping of the samples, and data interpretation was refined following a modified acacieae taxonomy. Application of the analytical strategy to investigate the paint binders in artworks from ancient Egypt allowed qualitative discrimination of gums at a species level, and provided new insights into the artists' material choices.

Assessment of monoclonal antibody glycosylation: a comparative study using HRMS, NMR, and HILIC-FLD.

Monoclonal antibodies (mAbs) represent the largest class of therapeutic protein drug products. mAb glycosylation produces a heterogeneous, analytically challenging distribution of glycoforms that typically should be adequately characterized because glycosylation-based product quality attributes (PQAs) can impact product quality, immunogenicity, and efficacy. In this study, two products were compared using a panel of analytical methods. Two high-resolution mass spectrometry (HRMS) workflows were used to analyze N-glycans, while nuclear magnetic resonance (NMR) was used to generate monosaccharide fingerprints. These state-of-the-art techniques were compared to conventional analysis using hydrophilic interaction chromatography (HILIC) coupled with fluorescence detection (FLD). The advantages and disadvantages of each method are discussed along with a comparison of the identified glycan distributions. The results demonstrated agreement across all methods for major glycoforms, demonstrating how confidence in glycan characterization is increased by combining orthogonal analytical methodologies. The full panel of methods used represents a diverse toolbox that can be selected from based on the needs for a specific product or analysis.

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.

Nutrient supplementation-induced metabolic profile changes and early appearance of free N-glycans in nutrient deficient tomato plants revealed by mass spectrometry.

Inorganic fertilizers are routinely used in large scale crop production for the supplementation of nitrogen, phosphorus, and potassium in nutrient poor soil. To explore metabolic changes in tomato plants grown on humic sand under different nutritional conditions, matrix-assisted laser desorption ionization (MALDI) mass spectrometry was utilized for the analysis of xylem sap. Variations in the abundances of metabolites and oligosaccharides, including free N-glycans (FNGs), were determined. Statistical analysis of the sample-related peaks revealed significant differences in the abundance ratios of multiple metabolites, including oligosaccharides, between the control plants, grown with no fertilizers, and plants raised under "ideal" and "nitrogen deficient" nutritional conditions, i.e., under the three treatment types. Among the 36 spectral features tentatively identified as oligosaccharides, the potential molecular structures for 18 species were predicted based on their accurate masses and isotope distribution patterns. To find the spectral features that account for most of the differences between the spectra corresponding to the three different treatments, multivariate statistical analysis was carried out by orthogonal partial least squares-discriminant analysis (OPLS-DA). They included both FNGs and non-FNG compounds that can be considered as early indicators of nutrient deficiency. Our results reveal that the potential nutrient deficiency indicators can be expanded to other metabolites beyond FNGs. The m/z values for 20 spectral features with the highest variable influence on projection (VIP) scores were ranked in the order of their influence on the statistical model.

Characterisation of a capsular polysaccharide from Moraxella nonliquefaciens CCUG 348T.

Moraxella nonliquefaciens is a commensal of the human upper respiratory tract (URT) but on rare occasions is recovered in cases of ocular, septic and pulmonary infections. Hence there is interest in the pathogenic determinants of M. nonliquefaciens, of which outer membrane (OM) structures such as fimbriae and two capsular polysaccharide (CPS) structures, →3)-ß-D-GalpNAc-(1→5)-ß-Kdop-(2→ and →8)-α-NeuAc-(2→, have been reported in the literature. To further characterise its surface virulence factors, we isolated a novel CPS from M. nonliquefaciens type strain CCUG 348T. This structure was elucidated using NMR data obtained from CPS samples that were subjected to various degrees of mild acid hydrolysis. Together with GLC-MS data, the structure was resolved as a linear polymer composed of two GalfNAc residues consecutively added to Kdo, →3)-ß-D-GalfNAc-(1→3)-α-D-GalfNAc-(1→5)-α-(8-OAc)Kdop-(2→. Supporting evidence for this material being CPS was drawn from the proposed CPS biosynthetic locus which encoded a potential GalfNAc transferase, a UDP-GalpNAc mutase for UDP-GalfNAc production and a putative CPS polymerase with predicted GalfNAc and Kdo transferase domains. This study describes a unique CPS composition reported in Moraxella spp. and offers genetic insights into the synthesis and expression of GalfNAc residues, which are rare in bacterial OM glycans.

Structure of the K141 capsular polysaccharide produced by Acinetobacter baumannii isolate KZ1106 that carries KL141 at the chromosomal K locus.

The structure of the K141 type capsular polysaccharide (CPS) produced by Acinetobacter baumannii KZ1106, a clinical isolate recovered from Kazakhstan in 2016, was established by sugar analyses and one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS was shown to consist of branched tetrasaccharide repeating units (K-units) with the following structure: This structure was found to be consistent with the genetic content of the KL141 CPS biosynthesis gene cluster at the chromosomal K locus in the KZ1106 whole genome sequence. Assignment of the encoded enzymes allowed the first sugar of the K unit to be identified, which revealed that the ß-d-GlcpNAc-(1→3)-d-GlcpNAc bond is the linkage between K-units formed by the WzyKL141 polymerase.

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.

Screening of polysaccharides from the differently processed products of Angelica sinensis with the best liver protection effect on chicken and the intervention mechanism study based on tandem mass tag proteomics and multiple reaction monitoring approach.

The incidence of colibacillosis in poultry is on the rise, significantly affecting the chicken industry. Ceftiofur sodium (CS) is frequently employed to treat this disease, resulting in lipopolysaccharide (LPS) buildup. Processing plays a vital role in traditional Chinese veterinary medicine. The potential intervention in liver injury by polysaccharides from the differently processed products of Angelica sinensis (PDPPAS) induced by combined CS and LPS remains unclear. This study aims to investigate the protective effect of PDPPAS on chicken liver injury caused by CS combined with LPS buildup and further identify the polysaccharides with the highest hepatoprotective activity in chickens. Furthermore, the study elucidates polysaccharides' intervention mechanism using tandem mass tag (TMT) proteomics and multiple reaction monitoring (MRM) methods. A total of 190 1-day-old layer chickens were randomly assigned into 12 groups, of which 14 chickens were in the control group and 16 in other groups, for a 10-day trial. The screening results showed that charred A. sinensis polysaccharide (CASP) had the most effective and the best hepatoprotective effect at 48 h. TMT proteomics and MRM validation results demonstrated that the intervention mechanism of the CASP high-dose (CASPH) intervention group was closely related to the protein expressions of FCER2, TBXAS1, CD34, AGXT, GCAT, COX7A2L, and CYP2AC1. Conclusively, the intervention mechanism of CASPH had multitarget, multicenter regulatory features.

Structure of rhamnoglucan, an unexpected alkali-stable polysaccharide extracted from Streptococcus mutans cell wall.

This study identified a rhamnose-containing cell wall polysaccharide (RhaCWP) in an alkaline extract prepared to analyze intracellular polysaccharides (IPS) from Streptococcus mutans biofilm. IPS was an 1,4-α-D-glucan with branchpoints introduced by 1,6-α-glucan while RhaCWP presented 1,2-α-L-and 1,3-α-L rhamnose backbone and side chains connected by 1,2-α-D-glucans, as identified by nuclear magnetic resonance (NMR) spectroscopy and methylation analyses. The MW of IPS and RhaCWP was 11,298 Da, as determined by diffusion-ordered NMR spectroscopy. Therefore, this study analyzed the chemical structure of RhaCWP and IPS from biofilm in a single fraction prepared via a convenient hot-alkali extraction method. This method could be a feasible approach to obtain such molecules and improve the comprehension of the structure-function relationships in polymers from S. mutans in future studies.

Extraction techniques, structural features and biological functions of Hippophae rhamnoides polysaccharides: A review.

Hippophae rhamnoides L. (sea buckthorn) is a type of traditional Chinese medicine with a long history of clinical application. It is used in the improvement and treatment of various diseases as medicine and food to strengthen the stomach and digestion, relieving cough and resolving phlegm, promoting blood circulation, and resolving blood stasis in traditional Chinese medicine. Emerging evidence has shown that H. rhamnoides polysaccharides (HRPs) are vital bioactive macromolecules responsible for its various health benefits. HRPs possess the huge potential to develop a drug improving or treating different diseases. In this review, we comprehensively and systematically summarize the recent information on extraction and purification methods, structural features, biological activities, structure-activity relationships, and potential industry applications of HRPs and further highlight the therapeutic potential and sanitarian functions of HRPs in the fields of therapeutic agents and functional food development. Additionally, this paper also lists a variety of biological activities of HRPs in vitro and in vivo roundly. Finally, this paper also discusses the structure-activity relationships and potential applications of HRPs. Overall, this work will help to have a better in-depth understanding of HRPs and provide a scientific basis and direct reference for more scientific and rational applications.

Onosma glomeratum Y. L. Liu polysaccharide alleviates LPS-induced pulmonary inflammation via NF-κB signal pathway.

As a traditional Chinese medicinal and edible homologous plant, Onosma glomeratum Y. L. Liu has been used for treating lung diseases in Tibet. In this study, a pectin polysaccharide, OGY-LLPA, with a molecular weight of 62,184 Da, was isolated and characterized by GC-MS and NMR analysis. It mainly consists of galacturonic acid (GalA), galactose (Gal), rhamnose (Rha), and arabinose (Ara), with a linear main chain of galacturonic acid (homogalacturonan, HG) inserted by part of rhamnose galacturonic acid (rhamnogalacturonan, RG), attaching with arabinogalactan (AG) branches at RG-I. Both in the LPS-induced A549 cell model and LPS-induced pneumonia mouse model, OGY-LLPA demonstrated strong anti-inflammatory effects, even comparable to DEX, indicating its potential as an anti-pneumonia candidate agent. Moreover, low-dose OGY-LLPA alleviated LPS-induced pulmonary inflammation by inhibiting the NF-κB signaling pathway. Overall, these findings could not only contribute to the utilization of Onosma glomeratum Y. L. Liu., but also provides a theoretical basis for the treatment of inflammation-related diseases.

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.