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Altered N -glycosylation of proteins on the cell membrane is associated with several neurodegenerative diseases. Microglia are an ideal model for studying glycosylation and neuroinflammation, but whether aberrant N -glycosylation in microglia can be restored by diet remains unknown. Herein, we profiled the N -glycome, proteome, and glycoproteome of the human microglia following lipopolysaccharide (LPS) induction to probe the impact of dietary and gut microbe-derived fatty acids-oleic acid, lauric acid, palmitic acid, valeric acid, butyric acid, isobutyric acid, and propionic acid-on neuroinflammation using liquid chromatography-tandem mass spectrometry. LPS changed N -glycosylation in the microglial glycocalyx altering high mannose and sialofucosylated N -glycans, suggesting the dysregulation of mannosidases, fucosyltransferases, and sialyltransferases. The results were consistent as we observed the restoration effect of the fatty acids, especially oleic acid, on the LPS-treated microglia, specifically on the high mannose and sialofucosylated glycoforms of translocon-associated proteins, SSRA and SSRB along with the cell surface proteins, CD63 and CD166. In addition, proteomic analysis and in silico modeling substantiated the potential of fatty acids in reverting the effects of LPS on microglial N -glycosylation. Our results showed that N -glycosylation is likely affected by diet by restoring alterations following LPS challenge, which may then influence the disease state.

Biological experiments are often conducted in vitro using immortalized cells due to their accessibility and ease of propagation compared to primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from the primary cell source due to the introduction of genes that enhance proliferation (e.g. CDK4) or enable telomere lengthening. To demonstrate the changes in phenotype upon CDK4-transformation, we performed LC-MS/MS glycomic and proteomic characterizations of a human lung cancer primary cell line (DTW75) and a CDK4-transformed cell line (GL01) derived from DTW75. We observed that the primary and CDK4-transformed cells expressed significantly different levels of sialylated, fucosylated, and sialofucosylated N-glycans. Specifically, the primary cells expressed higher levels of hybrid- and complex-type sialylated N-glycans, while CDK4-transformed cells expressed higher levels of complex-type fucosylated and sialofucosylated N-glycans. Further, we compared the proteomic differences between the cell lines and found that CDK4-transformed cells expressed higher levels of RNA-binding and adhesion proteins. Further, we observed that the CDK4-transformed cells changed N-glycosylation after 31 days in cell culture, with a decrease in high-mannose and increase in fucosylated, sialylated, and sialofucosylated N-glycans. Identifying these changes between primary and CDK4-transformed cells will provide useful insight when adapting cell lines that more closely resemble in vivo physiological conditions., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition 1-4 . Here we analyse biospecimens from a randomized, controlled trial of a microbiome-directed complementary food (MDCF-2) that produced superior rates of weight gain compared with a calorically more dense conventional ready-to-use supplementary food in 12-18-month-old Bangladeshi children with moderate acute malnutrition 4 . We reconstructed 1,000 bacterial genomes (metagenome-assembled genomes (MAGs)) from the faecal microbiomes of trial participants, identified 75 MAGs of which the abundances were positively associated with ponderal growth (change in weight-for-length Z score (WLZ)), characterized changes in MAG gene expression as a function of treatment type and WLZ response, and quantified carbohydrate structures in MDCF-2 and faeces. The results reveal that two Prevotella copri MAGs that are positively associated with WLZ are the principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilizing the component glycans of MDCF-2. The predicted specificities of carbohydrate-active enzymes expressed by their polysaccharide-utilization loci are correlated with (1) the in vitro growth of Bangladeshi P. copri strains, possessing varying degrees of polysaccharide-utilization loci and genomic conservation with these MAGs, in defined medium containing different purified glycans representative of those in MDCF-2, and (2) the levels of faecal carbohydrate structures in the trial participants. These associations suggest that identifying bioactive glycan structures in MDCFs metabolized by growth-associated bacterial taxa will help to guide recommendations about their use in children with acute malnutrition and enable the development of additional formulations., (© 2023. The Author(s).)

Glycomic profiling methods were used to determine the effect of metabolic inhibitors on glycan production. These inhibitors are commonly used to alter the cell surface glycosylation. However, structural analysis of the released glycans has been limited. In this research, the cell membranes were enriched and the glycans were released to obtain the N -glycans of the glycocalyx. Glycomic analysis using liquid chromatography-mass spectrometry (LC-MS) with a PGC chip column was used to profile the structures in the cell membrane. Glycans of untreated cells were compared to glycans of cells treated with inhibitors, including kifunensine, which inhibits the formation of complex- and hybrid-type structures, 2,4,7,8,9-Penta-O-acetyl- N -acetyl-3-fluoro-b-d-neuraminic acid methyl ester for sialylated glycans, 2-deoxy-2-fluorofucose, and 6-alkynyl fucose for fucosylated glycans. Kifunensine was the most effective, converting nearly 95% of glycans to high mannose types. The compound 6-alkynyl fucose inhibited some fucosylation but also incorporated into the glycan structure. Proteomic analysis of the enriched membrane for the four inhibitors showed only small changes in the proteome accompanied by large changes in the N -glycome for Caco-2. Future works may use these inhibitors to study the cellular behavior associated with the alteration of glycosylation in various biological systems, e.g., viral and bacterial infection, drug binding, and cell-cell interactions.

We have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the site-specific glycan alterations associated with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Glycopeptides associated with disease severity were also identified. Multinomial regression modelling was employed to construct and validate multi-analyte diagnostic models capable of accurately distinguishing PBC, PSC, and healthy controls from one another (AUC = 0.93 ± 0.03). Finally, to investigate how disease-relevant environmental factors can influence glycosylation, we characterized the ability of bile acids known to be differentially expressed in PBC to alter glycosylation. We hypothesize that this could be a mechanism by which altered self-antigens are generated and become targets for immune attack. This work demonstrates the utility of the MRM method to identify diagnostic site-specific glycan classifiers capable of distinguishing even related autoimmune diseases from one another., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.

Greater understanding of the spatial relationships between members of the human gut microbiota and available nutrients is needed to gain deeper insights about community dynamics and expressed functions. Therefore, we generated a panel of artificial food particles with each type composed of microscopic paramagnetic beads coated with a fluorescent barcode and one of 60 different dietary or host glycan preparations. Analysis of 160 Bacteroides and Parabacteroides strains disclosed diverse strain-specific and glycan-specific binding phenotypes. We identified carbohydrate structures that correlated with binding by specific bacterial strains in vitro and noted strain-specific differences in the catabolism of glycans that mediate adhesion. Mixed in vitro cultures revealed that these adhesion phenotypes are maintained in more complex communities. Additionally, orally administering glycan beads to gnotobiotic mice confirmed specificity in glycan binding. This approach should facilitate analyses of how strains occupying the same physical niche interact, and it should advance the development of synbiotics, more nutritious foods, and microbiota-based diagnostics., Competing Interests: Declaration of interests J.I.G. is a co-founder of Matatu, a company characterizing the role of diet-by-microbiota interactions in animal health. C.B.L. is the co-founder of Evolve Biosciences., (Copyright © 2021. Published by Elsevier Inc.)

N-glycosylation is a ubiquitous posttranslational modification that affects protein structure and function, including those of the central nervous system. N-glycans attached to cell membrane proteins play crucial roles in all aspects of biology, including embryogenesis, development, cell-cell recognition and adhesion, and cell signaling and communication. Although brain function and behavior are known to be regulated by the N-glycosylation state of numerous cell surface glycoproteins, our current understanding of brain glycosylation is limited, and glycan variations associated with functional brain regions remain largely unknown. In this work, we used a well-established cell surface glycomic nanoLC-Chip-Q-TOF platform developed in our laboratory to characterize the N-glycome of membrane fractions enriched in cell surface glycoproteins obtained from specific functional brain areas. We report the cell membrane N-glycome of two major developmental divisions of mice brain with specific and distinctive functions, namely the forebrain and hindbrain. Region-specific glycan maps were obtained with ∼120 N-glycan compositions in each region, revealing significant differences in "brain-type" glycans involving high mannose, bisecting, and core and antenna fucosylated species. Additionally, the cell membrane N-glycome of three functional regions of the forebrain and hindbrain, the cerebral cortex, hippocampus, and cerebellum, was characterized. In total, 125 N-glycan compositions were identified, and their region-specific expression profiles were characterized. Over 70 N-glycans contributed to the differentiation of the cerebral cortex, hippocampus, and cerebellum N-glycome, including bisecting and branched glycans with varying degrees of core and antenna fucosylation and sialylation. This study presents a comprehensive spatial distribution of the cell-membrane enriched N-glycomes associated with five discrete anatomical and functional brain areas, providing evidence for the presence of a previously unknown brain glyco-architecture. The region-specific molecular glyco fingerprints identified here will enable a better understanding of the critical biological roles that N-glycans play in the specialized functional brain areas in health and disease., Competing Interests: Conflict of 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 © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR < 0.05) site-specific inter-protein and intra-protein glycan associations, of which the vast majority were previously unknown. Since age and gender are relevant covariants in biomarker research, these variables were also characterized. 13 glycopeptides were found to be associated with gender and 41 to be associated with age. Using just five age-associated glycopeptides, a highly accurate age prediction model was constructed and validated (r 2  = 0.62 ± 0.12). The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions.

Polysaccharides are the most abundant biomolecules in nature, but are the least understood in terms of their chemical structures and biological functions. Polysaccharides cannot be simply sequenced because they are often highly branched and lack a uniform structure. Furthermore, large polymeric structures cannot be directly analyzed by mass spectrometry techniques, a problem that has been solved for polynucleotides and proteins. While restriction enzymes have advanced genomic analysis, and trypsin has advanced proteomic analysis, there has been no equivalent enzyme for universal polysaccharide digestion. We describe the development and application of a chemical method for producing oligosaccharides from polysaccharides. The released oligosaccharides are characterized by advanced liquid chromatography-mass spectrometry (LC-MS) methods with high sensitivity, accuracy and throughput. The technique is first used to identify polysaccharides by oligosaccharide fingerprinting. Next, the polysaccharide compositions of food and feces are determined, further illustrating the utility of technique in food and clinical studies.

Proteins on cell membrane are modified by N- and O-glycans. N-Glycans have been extensively characterized using advanced separation and mass spectrometry techniques. However, O-glycans remain a challenge, because of the lack of universal enzymes to release them and the large background abundances of N-glycans. Here, we report a method for in-depth structural analysis and quantitation of O-glycans derived from human cell membrane. O-Glycans were chemically released from isolated cell membrane glycoproteins following N-glycan and lipid/glycolipid removal by PNGase F digestion and Folch extraction, respectively. Released O-glycans were purified by an optimized protocol to eliminate interference from small molecules and degraded proteins. Cell surface O-glycans were then analyzed using a nanoLC-chip-QTOF mass spectrometer with a porous graphitized carbon (PGC) column, while the N-glycans and glycolipids isolated from the same cell membrane fractions were analyzed in parallel using previously reported methods. The monosaccharide compositions and linkages of the detected O-glycans were identified by exoglycosidase digestion facilitated with tandem mass spectrometry (MS/MS). Using this method, we identified 44 cell membrane O-glycan isomers with MS/MS, and, among them, we unambiguously characterized 25 O-glycan structures with exoglycosidase digestion to create a library with their complete structures, accurate masses, and retention times. In this process, we identified and characterized unexpected mannose oligomers that are α(1-2/3) linked. This library enabled the identification and quantification of unique cell surface O-glycans from different cell lines and the study of specific O-glycan changes during cell differentiation.

Secretory Immunoglobulin A (SIgA) is central to mucosal immunity: represents one of the main immunological mechanisms of defense against the potential attack of pathogens. During lactation SIgA is produced by plasmablasts in the mammary gland and is present in breast milk, playing a vital role in the passive immunity of the newborn. Interestingly, the different components of SIgA are highly N-glycosylated, and these N-Glycans have an essential role in health maintenance. In this work, we performed a glycomic study to compare N-glycosylation of SIgA purified from mature breast milk and saliva, and plasma IgA from the same lactating participants. Our results revealed a greater diversity than previously reported, with 89 glycan compositions that may correspond to over 250 structures. Among these glycans, 54 glycan compositions were characterized as body-fluid specific. Most of these unique N-Glycan compositions identified in SIgA from mature milk and IgA from plasma were fucosylated and both fucosylated and sialylated species, whereas in salivary SIgA the unique structures were mainly undecorated complex N-Glycans. In addition, we evaluated the effect of delivery mode on (S)IgA glycosylation. Lactating participants who had given birth by vaginal delivery presented an increased proportion of high mannose and fucosylated glycans in salivary SIgA, and selected high mannose, fucosylated, sialylated, and both fucosylated and sialylated glycans in plasma IgA, indicating that the hormonal changes during vaginal delivery could affect plasma and saliva IgA. These results reveal the structural details that provide a new dimension to the roles of (S)IgA N-Glycans in different tissues, and especially in maternal and new-born protection and infant development. The design of optimal recombinant IgA molecules specifically targeted to protect mucosal surfaces will need to include this dimension of structural detail., (© 2019 Goonatilleke et al.)

The recruitment of a bacterial consortium by the host is a strategy not limited to animals but is also used in plants. A maize aerial root mucilage has been found that harbors nitrogen fixing bacteria that are attracted to the carbohydrate rich environment. This synbiotic relationship is facilitated by a polysaccharide, whose complicated structure has been previously unknown. In this report, we present the characterization of the maize polysaccharide by employing new analytical strategies combining chemical depolymerization, oligosaccharide sequencing, and monosaccharide and glycosidic linkage quantitation. The mucilage contains a single heterogeneous polysaccharide composed of a highly fucosylated and xylosylated galactose backbone with arabinan and mannoglucuronan branches. This unique polysaccharide structure may select for the diazotrophic community by containing monosaccharides and linkages that correspond to the glycosyl hydrolases associated with the microbial community. The elucidation of this complicated structure illustrates the power of the analytical methods, which may serve as a general platform for polysaccharide analysis in the future.

The structural analysis of carbohydrates remains challenging mainly due to the lack of rapid analytical methods able to determine and quantitate glycosidic linkages between the diverse monosaccharides found in natural oligosaccharides and polysaccharides. In this research, we present the first liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the rapid and simultaneous relative quantitation of glycosidic linkages for oligosaccharide and polysaccharide characterization. The method developed employs ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ-MS) analysis performed in multiple reaction monitoring (MRM) mode. A library of 22 glycosidic linkages was built using commercial oligosaccharide standards. Permethylation and hydrolysis conditions along with LC-MS/MS parameters were optimized resulting in a workflow requiring only 50 μg of substrate for the analysis. Samples were homogenized, permethylated, hydrolyzed, and then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) prior to analysis by UHPLC/MRM-MS. Separation by C18 reversed-phase UHPLC along with the simultaneous monitoring of derivatized terminal, linear, bisecting, and trisecting monosaccharide linkages by mass spectrometry is achieved within a 15 min run time. Reproducibility, efficacy, and robustness of the method was demonstrated with galactan ( Lupin) and polysaccharides within food such as whole carrots. The speed and specificity of the method enables its application toward the rapid glycosidic linkage analysis of oligosaccharides and polysaccharides.

Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycans are produced through a complicated nontemplate driven process involving the competition of enzymes that extend the nascent chain. The large diversity of structures, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies of glycans all conspire to make the analysis arguably much more difficult than any other biopolymer. Furthermore, the large number of glycoforms associated with a specific protein site makes it more difficult to characterize than any post-translational modification. Nonetheless, there have been significant progress, and advanced separation and mass spectrometry methods have been at its center and the main reason for the progress. While glycomic and glycoproteomic analyses are still typically available only through highly specialized laboratories, new software and workflow is making it more accessible. This review focuses on the role of mass spectrometry and separation methods in advancing glycomic and glycoproteomic analyses. It describes the current state of the field and progress toward making it more available to the larger scientific community.

Since hundreds of clinical trials are investigating the use of multipotent stromal cells (MSCs) for therapeutic purposes, effective delivery of the cells to target tissues is critical. We have found an unexplored mechanism, by which basic fibroblast growth factor (FGF2) induces expression of fucosyltransferase 8 (FUT8) to increase core fucosylations of N-linked glycans of membrane-associated proteins, including several integrin subunits. Gain- and loss-of-function experiments show that FUT8 is both necessary and sufficient to induce migration of MSCs. Silencing FUT8 also affects migration of MSCs in zebrafish embryos and a murine bone fracture model. Finally, we use in silico modeling to show that core fucosylations restrict the degrees of freedom of glycans on the integrin's surface, hence stabilizing glycans on a specific position. Altogether, we show a mechanism whereby FGF2 promotes migration of MSCs by modifying N-glycans. This work may help improve delivery of MSCs in therapeutic settings., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glycosphingolipids (GSLs) are found in cellular membranes of most organisms and play important roles in cell-cell recognition, signaling, growth, and adhesion, among others. A method based on nanoflow high performance liquid chromatography-chip-quadrupole-time-of-flight mass spectrometry (nanoHPLC Chip-Q-TOF MS) was applied towards identifying and quantifying intact GSLs from a variety of samples, including cultured cell lines and animal tissue. The method provides the composition and sequence of the glycan, as well as variations in the ceramide portion of the GSL. It was used to profile the changes in the glycolipidome of Caco-2 cells as they undergo differentiation. A total of 226 unique GSLs were found among Caco-2 samples from five differentiation time-points. The method provided a comprehensive glycolipidomic profile of a cell during differentiation to yield the dynamic variation of intact GSL structures.

Osteonecrosis of the femoral head is a recalcitrant and paralyzing disease often discovered in the end stage at the time of diagnosis, which is often performed by physical examination and diagnostic imaging. Osteonecrosis of the femoral head is typically caused by trauma or long-term steroid use. There are over 30 million patients in the US taking steroids, and roughly 40% will develop osteonecrosis of the femoral head. However, the exact pathophysiological process is not well understood. This study aims to examine the alteration in serum glycosylation of osteonecrosis of the femoral head using the state-of-the-art analytical tools to provide more chemical data for pathophysiology research and possibly biomarker discovery. A training set containing 27 serum samples from steroid-induced osteonecrosis of the femoral head patients and 25 from gender- and age-matched controls was collected and analyzed. Glycosylation of whole serum and site-specific glycosylation of immunoglobulins are characterized using electrospray ionization-Q-time of flight and electrospray ionization-Triple-Quadruple via multiple reaction monitoring, respectively. The whole serum glycosylation analysis yielded 14 N-glycan compositions and multiple reaction monitoring yielded eight glycopeptides that were altered between cases and controls with statistical significance. The increase of nonsialylated, nonfucosylated N-glycans and decrease of fucosylated N-glycans are associated with the development of osteonecrosis of the femoral head. Glycosylation is a posttranslational protein modification and is apparently affected by osteonecrosis of the femoral head. Future studies with a larger cohort and patients from earlier stage will be performed to assess these potential markers' value in disease onset.

Protein glycosylation and other post-translational modifications are involved in potentially all aspects of human growth and development. Defective glycosylation has adverse effects on human physiological conditions and accompanies many chronic and infectious diseases. Altered glycosylation can occur at the onset and/or during tumor progression. Identifying these changes at early disease stages may aid in making decisions regarding treatments, as early intervention can greatly enhance survival. This review highlights some of the efforts being made to identify N- and O-glycosylation profile shifts in cancer using mass spectrometry. The analysis of single or panels of potential glycoprotein cancer markers are covered. Other emerging technologies such as global glycan release and site-specific glycosylation analysis and quantitation are also discussed. Graphical Abstract Steps involved in the biomarker discovery., Competing Interests: The authors have declared no conflict of interest.

A new acridone derivative 2-aminoacetamido-10-(3, 5-dimethoxy)-benzyl-9(10H)-acridone hydrochloride (8a) has been shown to have potent antitumor activity. In order to understand the underlying action mechanism of 8a, three compounds of the same class with structures optimized step-by-step, 9(10H)-acridone (A), 10-(3,5-dimethoxy) benzyl-9(10H)-acridone (I) and 8a, were exposed to CCRF-CEM leukemia cell to determine the N-glycosylation changes using the microfluidic HPLC-chip-TOF MS platform. N-Glycans from whole cell lysates (WCL) and cell membranes (CM) were analyzed using isomer-sensitive chip-based porous graphitized carbon nano-LC/MS. A total of 223 N-glycan compositions and 398 N-glycan compounds were identified. Comparison of the two analyses showed that more apparent changes were observed in the CM compared with WCL, suggesting that CM may be a more sensitive indicator of changes in glycosylation. Upon 8a exposure to CCRF-CEM cells, a significant decrease in high-mannose-type glycans was observed. Different expressions of oligosaccharyltransferase subunits appear to play a key functional role in regulating the hypoglycosylation and contribute to the action mechanism of 8a. Taken together our findings suggest that glycosylation is strongly affected by therapeutic potency and can be used as possible biomarkers for monitoring toxicity and antitumor activity of 8a., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although gut host-pathogen interactions are glycan-mediated processes, few details are known about the participating structures. Here we employ high-resolution mass spectrometric profiling to comprehensively identify and quantitatively measure the exact modifications of native intestinal epithelial cell surface N-glycans induced by S. typhimurium infection. Sixty minutes postinfection, select sialylated structures showed decreases in terms of total number and abundances. To assess the effect of cell surface mannosylation, we selectively rerouted glycan expression on the host using the alpha-mannosidase inhibitor, kifunensine, toward overexpression of high mannose. Under these conditions, internalization of S. typhimurium significantly increased, demonstrating that bacteria show preference for particular structures. Finally, we developed a novel assay to measure membrane glycoprotein turnover rates, which revealed that glycan modifications occur by bacterial enzyme activity rather than by host-derived restructuring strategies. This study is the first to provide precise structural information on how host N-glycans are altered to support S. typhimurium invasion., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Complex glycans cover the gut epithelial surface to protect the cell from the environment. Invasive pathogens must breach the glycan layer before initiating infection. While glycan degradation is crucial for infection, this process is inadequately understood. Salmonella contains 47 glycosyl hydrolases (GHs) that may degrade the glycan. We hypothesized that keystone genes from the entire GH complement of Salmonella are required to degrade glycans to change infection. This study determined that GHs recognize the terminal monosaccharides (N-acetylneuraminic acid (Neu5Ac), galactose, mannose, and fucose) and significantly (p < 0.05) alter infection. During infection, Salmonella used its two GHs sialidase nanH and amylase malS for internalization by targeting different glycan structures. The host glycans were altered during Salmonella association via the induction of N-glycan biosynthesis pathways leading to modification of host glycans by increasing fucosylation and mannose content, while decreasing sialylation. Gene expression analysis indicated that the host cell responded by regulating more than 50 genes resulting in remodeled glycans in response to Salmonella treatment. This study established the glycan structures on colonic epithelial cells, determined that Salmonella required two keystone GHs for internalization, and left remodeled host glycans as a result of infection. These data indicate that microbial GHs are undiscovered virulence factors.

Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung cancer. Here, we aimed to determine the predictive value of serum glycans to distinguish non-small cell lung cancer (NSCLC) cases from controls in prediagnostic samples using a previously validated predictive protein marker pro-SFTPB, as anchor. Blinded prediagnostic serum samples were obtained from the Carotene and Retinol Efficacy Trial (CARET), and included a discovery set of 100 NSCLC cases and 199 healthy controls. A second test set consisted of 108 cases and 216 controls. Cases and controls were matched for age at baseline (5-year groups), sex, smoking status (current vs. former), study enrollment cohort, and date of blood draw. Serum glycan profiles were determined by mass spectrometry. Twelve glycan variables were identified to have significant discriminatory power between cases and controls in the discovery set (AUC > 0.6). Of these, four were confirmed in the independent validation set. A combination marker yielded AUCs of 0.74 and 0.64 in the discovery and test set, respectively. Four glycan variables exhibited significant incremental value when combined with pro-SFTPB compared with pro-SFTPB alone with AUCs of 0.73, 0.72, 0.72, and 0.72 in the test set, indicating that serum glycan signatures have relevance to risk assessment for NSCLC., (©2016 American Association for Cancer Research.)

To decrease the mortality of lung cancer, better screening and diagnostic tools as well as treatment options are needed. Protein glycosylation is one of the major post-translational modifications that is altered in cancer, but it is not exactly clear which glycan structures are affected. A better understanding of the glycan structures that are differentially regulated in lung tumor tissue is highly desirable and will allow us to gain greater insight into the underlying biological mechanisms of aberrant glycosylation in lung cancer. Here, we assess differential glycosylation patterns of lung tumor tissue and nonmalignant tissue at the level of individual glycan structures using nLC-chip-TOF-MS. Using tissue samples from 42 lung adenocarcinoma patients, 29 differentially expressed (FDR < 0.05) glycan structures were identified. The levels of several oligomannose type glycans were upregulated in tumor tissue. Furthermore, levels of fully galactosylated glycans, some of which were of the hybrid type and mostly without fucose, were decreased in cancerous tissue, whereas levels of non- or low-galactosylated glycans mostly with fucose were increased. To further assess the regulation of the altered glycosylation, the glycomics data was compared to publicly available gene expression data from lung adenocarcinoma tissue compared to nonmalignant lung tissue. The results are consistent with the possibility that the observed N-glycan changes have their origin in differentially expressed glycosyltransferases. These results will be used as a starting point for the further development of clinical glycan applications in the fields of imaging, drug targeting, and biomarkers for lung cancer.

Glycosylation is an important post-translational modification of proteins present in the vast majority of human proteins. For this reason, they are potentially new sources of biomarkers and active targets of therapeutics and vaccines. However, the absence of a biosynthetic template as in the genome and the general complexity of the structures have limited the development of methods for comprehensive structural analysis. Even now, the exact structures of many abundant N-glycans in serum are not known. Structural elucidation of oligosaccharides remains difficult and time-consuming. Here, we introduce a means of rapidly identifying released N-glycan structures using their accurate masses and retention times based on a glycan library. This serum glycan library, significantly expanded from a previous one covering glycans released from a handful of serum glycoproteins, has more than 170 complete and partial structures and constructed instead from whole serum. The method employs primarily nanoflow liquid chromatography and accurate mass spectrometry. The method allows us to readily profile N-glycans in biological fluids with deep structural analysis. This approach is used to determine the relative abundances and variations in the N-glycans from several individuals providing detailed variations in the abundances of the important N-glycans in blood.

Herein we will review the role of glycans in the immune system. Specific topics covered include: the glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode "self" identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migration pattern of immune cells; and how the combination of Fc N-glycans and Ig isotype dictate the effector function of immunoglobulins. We speculate that the latter may be responsible for the well-documented association between alterations of the serum glycome and autoimmunity. Due to technological limitations, the extent of these autoimmune-associated glycan alterations and their role in disease pathophysiology has not been fully elucidated. Thus, we also review the current technologies available for glycan analysis, placing an emphasis on Multiple Reaction Monitoring (MRM), a rapid high-throughput technology that has great potential for glycan biomarker research. Finally, we put forth The Altered Glycan Theory of Autoimmunity, which states that each autoimmune disease will have a unique glycan signature characterized by the site-specific relative abundances of individual glycan structures on immune cells and extracellular proteins, especially the site-specific glycosylation patterns of the different immunoglobulin(Ig) classes and subclasses., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An improved understanding of glycosylation will provide new insights into many biological processes. In the analysis of oligosaccharides from biological samples, a strict regime is typically followed to ensure sample integrity. However, the fate of glycans that have been exposed to environmental conditions over millennia has not yet been investigated. This is also true for understanding the evolution of the glycosylation machinery in humans as well as in any other biological systems. In this study, we examined the glycosylation of tissue samples derived from four mummies which have been naturally preserved: - the 5,300 year old "Iceman called Oetzi", found in the Tyrolean Alps; the 2,400 year old "Scythian warrior" and "Scythian Princess", found in the Altai Mountains; and a 4 year old apartment mummy, found in Vienna/Austria. The number of N-glycans that were identified varied both with the age and the preservation status of the mummies. More glycan structures were discovered in the contemporary sample, as expected, however it is significant that glycan still exists in the ancient tissue samples. This discovery clearly shows that glycans persist for thousands of years, and these samples provide a vital insight into ancient glycosylation, offering us a window into the distant past.

In clinical settings, biopsies are routinely used to determine cancer type and grade based on tumor cell morphology, as determined via histochemical or immunohistochemical staining. Unfortunately, in a significant number of cases, traditional biopsy results are either inconclusive or do not provide full subtype differentiation, possibly leading to inefficient or ineffective treatment. Glycomic profiling of the cell membrane offers an alternate route toward cancer diagnosis. In this study, isomer-sensitive nano-LC/MS was used to directly obtain detailed profiles of the different N-glycan structures present on cancer cell membranes. Membrane N-glycans were extracted from cells representing various subtypes of breast, lung, cervical, ovarian, and lymphatic cancer. Chip-based porous graphitized carbon nano-LC/MS was used to separate, identify, and quantify the native N-glycans. Structure-sensitive N-glycan profiling identified hundreds of glycan peaks per cell line, including multiple isomers for most compositions. Hierarchical clusterings based on Pearson correlation coefficients were used to quickly compare and separate each cell line according to originating organ and disease subtype. Based simply on the relative abundances of broad glycan classes (e.g., high mannose, complex/hybrid fucosylated, complex/hybrid sialylated, etc.), most cell lines were readily differentiated. More closely related cell lines were differentiated based on several-fold differences in the abundances of individual glycans. Based on characteristic N-glycan profiles, primary cancer origins and molecular subtypes could be distinguished. These results demonstrate that stark differences in cancer cell membrane glycosylation can be exploited to create an MS-based biopsy, with potential applications toward cancer diagnosis and direction of treatment.

Glycomics, a comprehensive study of glycans expressed in biologic systems, is emerging as a simple yet highly sensitive diagnostic tool for disease onset and progression. This study aimed to use glycomics to investigate glycan markers that would differentiate patients with gastric cancer from those with nonatrophic gastritis. Patients with duodenal ulcer were also included because they are thought to represent a biologically different response to infection with Helicobacter pylori, a bacterial infection that can cause either gastric cancer or duodenal ulcer. We collected 72 serum samples from patients in Mexico City that presented with nonatrophic gastritis, duodenal ulcer, or gastric cancer. N-glycans were released from serum samples using the generic method with PNGase F and were analyzed by matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry. The corresponding glycan compositions were calculated based on accurate mass. ANOVA-based statistical analysis was performed to identify potential markers for each subgroup. Nineteen glycans were significantly different among the diagnostic groups. Generally, decreased levels of high-mannose-type glycans, glycans with one complex type antenna, bigalactosylated biantennary glycans, and increased levels of nongalactosylated biantennary glycans were observed in gastric cancer cases. Altered levels of serum glycans were also observed in duodenal ulcer, but differences were generally in the same direction as gastric cancer. Serum glycan profiles may provide biomarkers to differentiate gastric cancer cases from controls with nonatrophic gastritis. Further studies will be needed to validate these findings as biomarkers and identify the role of protein glycosylation in gastric cancer pathology.

Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits further progress in the areas of proteomics and glycomics. Effective methods require new approaches in sample preparation, detection, and data analysis. While the field has advanced in sample preparation and detection, automated data analysis remains an important goal. A new bioinformatics approach implemented in software called GP Finder automatically distinguishes correct assignments from random matches and complements experimental techniques that are optimal for glycopeptides, including nonspecific proteolysis and high mass resolution liquid chromatography/tandem mass spectrometry (LC/MS/MS). SSG for multiple N- and O-glycosylation sites, including extensive glycan heterogeneity, was annotated for single proteins and protein mixtures with a 5% false-discovery rate, generating hundreds of nonrandom glycopeptide matches and demonstrating the proof-of-concept for a self-consistency scoring algorithm shown to be compliant with the target-decoy approach (TDA). The approach was further applied to a mixture of N-glycoproteins from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein (vLDL) particles.

Mice are the premier mammalian models for studies of human physiology and disease, bearing extensive biological similarity to humans with far fewer ethical, economic, or logistic complications. To facilitate glycomic studies based on the mouse model, we comprehensively profiled the mouse serum N-glycome using isomer-specific nano-LC/MS and -LC/MS/MS. N-Glycans were identified by accurate mass MS and structurally elucidated by MS/MS. Porous graphitized carbon nano-LC was able to separate out nearly 300 N-linked glycan compounds (including isomers) from just over 100 distinct N-linked glycan compositions. Additional MS/MS structural analysis was performed on a number of novel N-glycans, revealing the structural characteristics of modifications such as dehydration, O-acetylation, and lactylation. Experimental findings were combined with known glycobiology to generate a theoretical library of all biologically possible mouse serum N-glycan compositions. The library may be used for automated identification of complex mixtures of mouse N-glycans, with possible applications to a wide range of mouse-related research endeavors, including pharmaceutical drug development and biomarker discovery.

Many studies focused on the discovery of novel biomarkers for the diagnosis and treatment of disease states are facilitated by mass spectrometry-based technology. HPLC coupled to mass spectrometry is widely used; miniaturization of this technique using nano-liquid chromatography (LC)-mass spectrometry (MS) usually results in better sensitivity, but is associated with limited repeatability. The recent introduction of chip-based technology has significantly improved the stability of nano-LC-MS, but no substantial studies to verify this have been performed. To evaluate the temporal repeatability of chip-based nano-LC-MS analyses, N-glycans released from a serum sample were repeatedly analyzed using nLC-PGC-chip-TOF-MS on three non-consecutive days. With an average inter-day coefficient of variation of 4 %, determined on log10-transformed integrals, the repeatability of the system is very high. Overall, chip-based nano-LC-MS appears to be a highly stable technology, which is suitable for the profiling of large numbers of clinical samples for biomarker discovery.

O-Glycans in saliva and tear isolated from patients suffering from ocular rosacea, a form of inflammatory ocular surface disease, were profiled, and their structures were elucidated using high resolution mass spectrometry. We have previously shown that certain structures, particularly sulfated oligosaccharides, increased in the tear and saliva of rosacea patients. In this study, the structures of these glycans were elucidated using primarily tandem mass spectrometry. There were important similarities in the glycan profiles of tears and saliva with the majority of the structures in common. The structures of the most abundant species common to both tear and saliva, which were also the most abundant species in both, were elucidated. For sulfated species, the positions of the sulfate groups were localized. The majority of the structures were new, with the sulfated glycans comprising mucin core 1- and core 2-type structures. As both saliva and tear are rich in mucins, it is suggested that the O-glycans are mainly components of mucins. The study further illustrates the strong correspondence between the glycans in the tear and saliva of ocular rosacea patients.

Aberrant glycosylation has been observed for decades in essentially all types of cancer, and is now well established as an indicator of carcinogenesis. Mining the glycome for biomarkers, however, requires analytical methods that can rapidly separate, identify, and quantify isomeric glycans. We have developed a rapid-throughput method for chromatographic glycan profiling using microfluidic chip-based nanoflow liquid chromatography (nano-LC)/mass spectrometry. To demonstrate the utility of this method, we analyzed and compared serum samples from epithelial ovarian cancer cases (n=46) and healthy control individuals (n=48). Over 250 N-linked glycan compound peaks with over 100 distinct N-linked glycan compositions were identified. Statistical testing identified 26 potential glycan biomarkers based on both compositional and structure-specific analyses. Using these results, an optimized model was created incorporating the combined abundances of seven potential glycan biomarkers. The receiver operating characteristic (ROC) curve of this optimized model had an area under the curve (AUC) of 0.96, indicating robust discrimination between cancer cases and healthy controls. Rapid-throughput chromatographic glycan profiling was found to be an effective platform for structure-specific biomarker discovery., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO). To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.

Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-β-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.

Human serum glycomics is a promising method for finding cancer biomarkers but often lacks the tools for streamlined data analysis. The Glycolyzer software incorporates a suite of analytic tools capable of identifying informative glycan peaks out of raw mass spectrometry data. As a demonstration of its utility, the program was used to identify putative biomarkers for epithelial ovarian cancer from a human serum sample set. A randomized, blocked, and blinded experimental design was used on a discovery set consisting of 46 cases and 48 controls. Retrosynthetic glycan libraries were used for data analysis and several significant candidate glycan biomarkers were discovered via hypothesis testing. The significant glycans were attributed to a glycan family based on glycan composition relationships and incorporated into a linear classifier motif test. The motif test was then applied to the discovery set to evaluate the disease state discrimination performance. The test provided strongly predictive results based on receiver operator characteristic curve analysis. The area under the receiver operator characteristic curve was 0.93. Using the Glycolyzer software, we were able to identify a set of glycan biomarkers that highly discriminate between cases and controls, and are ready to be formally validated in subsequent studies., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The purpose of this study was to study changes in glycosylation in tear and saliva obtained from control and ocular rosacea patients in order to identify potential biomarkers for rosacea. Tear fluid was collected from 51 subjects (28 healthy controls and 23 patients with ocular rosacea). Saliva was collected from 42 of the same subjects (25 controls and 17 patients). Pooled and individual samples were examined to determine overall glycan profiles and individual variations in glycosylation. O-and N- glycans were released from both patients and control subjects. Released glycans were purified and enriched by solid-phase extraction (SPE) with graphitized carbon. Glycans were eluted based on glycan size and polarity. SPE fractions were then analyzed by high-resolution mass spectrometry. Glycan compositions were assigned by accurate masses. Their structures were further elucidated by tandem mass spectrometric using collision-induced dissociation (CID), and specific linkage information was obtained by exoglycosidase digestion. N- and O-glycans were released from 20-μL samples without protein identification, separation, and purification. Approximately 50 N-glycans and 70 O-glycans were globally profiled by mass spectrometry. Most N-glycans were highly fucosylated, while O-glycans were sulfated. Normal tear fluid and saliva contain highly fucosylated glycans. The numbers of sulfated glycans were dramatically increased in tear and saliva of rosacea patients compared to controls. Glycans found in tear and saliva from roseatic patients present highly quantitative similarity. The abundance of highly fucosylated N-glycans in the control samples and sulfated O-glycans in ocular rosacea patient samples may lead to the discovery of an objective diagnostic marker for the disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation.

Glycosylation is one of the most common post-translational modifications of proteins and has been shown to change with various pathological states including cancer. Global glycan profiling of human serum based on mass spectrometry has already led to several promising markers for diseases. The changes in glycan structure can result in altered monosaccharide composition as well as in the linkages between the monosaccharides. High-throughput glycan structural elucidation is not possible because of the lack of a glycan template to expedite identification. In an effort toward rapid profiling and identification of glycans, we have constructed a library of structures for the serum glycome to aid in the rapid identification of serum glycans. N-Glycans from human serum glycoproteins are used as a standard and compiled into a library with exact structure (composition and linkage), liquid chromatography retention time, and accurate mass. Development of the library relies on highly reproducible nanoLC-MS retention times. Tandem MS and exoglycosidase digestions were used for structural elucidation. The library currently contains over 300 entries with 50 structures completely elucidated and over 60 partially elucidated structures. This database is steadily growing and will be used to rapidly identify glycans in unknown biological samples.

Serum N-glycan profiles for use as clinical biomarkers of disease(s) is of increasing scientific interest. Promising profiles have already been identified in several diseases, including cancer, Alzheimer's, and diabetes. Venipuncture is routinely performed to collect the blood necessary for this type of analysis, but blood from a fingerstick placed on filter paper (known as dried blood spots (DBS)) is more advantageous. This sampling method is less invasive than "classical" blood drawing, can be performed conveniently at home, and avoids cumbersome shipping and storage procedures. Here, we present a procedure for N-glycan profiling of DBS samples consisting of reconstitution of DBS in N-glycan release buffer, protein denaturation, enzymatic N-glycan release and PGC Solid phase extraction (SPE) for purification. Samples are then analyzed using nanoHPLC-PGC-chip-TOF-MS to generate N-glycan profiles. Using this method, ~150 N-glycan structures can be monitored, originating from 44 N-glycan compositions that can be analyzed with good repeatability (the coefficient of variation (%CV) is ~20%). To assess the stability of the N-glycans during storage, DBS samples were stored at room temperature (RT) and -80 °C. No major differences in N-glycan composition could be observed. Moreover, upon comparison of the N-glycan profile of DBS with profiles obtained from serum, which is a classical matrix for N-glycan profiling, similar patterns were observed. The method facilitates large population studies for N-glycan profiling, and is especially advantageous for children and the elderly, who have limited blood supplies, as well as animal studies in small mammals., (© 2011 American Chemical Society)

Glycosylation is highly sensitive to the biochemical environment and has been implicated in many diseases including cancer. Glycan compositional profiling of human serum with mass spectrometry has already identified potential biomarkers for several types of cancer and diseases; however, composition alone does not fully describe glycan stereo- and regioisomeric diversity. The vast structural heterogeneity of glycans presents a formidable analytical challenge. We have developed a method to identify and quantify isomeric native glycans using nanoflow liquid chromatography (nano-LC)/mass spectrometry. A microfluidic chip packed with graphitized carbon was used to chromatographically separate the glycans. To determine the utility of this method for structure-specific biomarker discovery, we analyzed serum samples from two groups of prostate cancer patients with different prognoses. More than 300 N-glycan species (including isomeric structures) were identified, corresponding to over 100 N-glycan compositions. Statistical tests established significant differences in glycan abundances between patient groups. This method provides comprehensive, selective, and quantitative glycan profiling.

Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ∼30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.

N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.

Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. We observed four N-linked glycans, m/z 1339.480 (Hex(3)HexNAc), 1485.530 (Hex(3)HexNAc(4)Fuc), 1809.639 (Hex(5)HexNAc(4)Fuc), and 1905.630 (Man(9)), change in intensity in the cancer group but not in the control group. In a separate study, N-glycans from total human serum glycoproteins of breast cancer patients and controls were also profiled. Analysis of human sera using an internal standard showed the alteration of the low-abundant high-mannose glycans, m/z 1419.475, 1581.528, 1743.581, 1905.634 (Man(6-9)), in breast cancer patients. A key observation was the elevation of a high-mannose type glycan containing nine mannoses, Man(9), m/z 1905.630 in both mouse and human sera in the presence of breast cancer, suggesting an incompletion of the glycosylation process that normally trims back Man(9) to produce complex and hybrid type oligosaccharides.

Glycans constitute a new class of compounds for biomarker discovery. Glycosylation is a common post-translational modification and is often associated with transformation to malignancy. To analyze glycans, they are released from proteins, enriched, and measured with mass spectrometry. For biomarker discovery, repeatability at every step of the process is important. Locating and minimizing the process variability is key to establishing a robust platform stable enough for biomarker discovery. Understanding the variability of the measurement devices helps understand the variability associated with the chemical processing. This report explores the potential use of methods expediting the enzymatic release of glycans such as a microwave reactor and automation of the solid-phase extraction with a robotic liquid handler. The study employs matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry but would be suitable with any mass spectrometry method. Methods for system-wide data analysis are examined because proper metrics for evaluating the performance of glycan sample preparation procedures are not well established.

Glycosylation is one of the most common post-translational modifications (PTMs) of proteins. At least 50% of human proteins are glycosylated with some estimates being as high as 70%. Glycoprotein analysis requires determining both the sites of glycosylation as well as the glycan structures associated with each site. Recent advances have led to the development of new analytical methods that employ mass spectrometry extensively making it possible to obtain the glycosylation site and the site microheterogeneity. These tools will be important for the eventual development of glycoproteomics.