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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.

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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

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.

Cell membrane protein glycosylation is dependent on the metabolic state of the cell as well as exogenous nutrients available. Although the metabolism and interconversion of monosaccharides have been well-studied, their incorporation into cell surface glycans and their corresponding glycoproteins remains relatively unknown. In this study, we developed a method to investigate quantitatively the incorporation pathways of dietary saccharides into specific glycans and glycoproteins on the cell membrane by treating intestinal Caco-2 and hepatic KKU-M213 cells with 13C-labeled monosaccharides and characterizing the resulting cell surface glycans and glycopeptides by LC-MS/MS. Time-course studies using uniformly labeled glucose revealed that the rate of incorporation was both glycan-specific and protein-dependent. Comparative studies using different dietary saccharides and multiple cell lines revealed the variance of monosaccharide utilization and interconversion in different tissues and organisms. The robust isotope-labeling and glycan profiling methods can provide a useful tool for differentiating glycosylation pathways and enhance the understanding of how dietary sugar intake affects health.

O-GlcNAcylation is a key post-translational modification that modifies the functions of proteins. Associations between O-GlcNAcylation, shorter survival of cholangiocarcinoma (CCA) patients, and increased migration/invasion of CCA cell lines have been reported. However, the specific O-GlcNAcylated proteins (OGPs) that participate in promotion of CCA progression are poorly understood. OGPs were isolated from human CCA cell lines, KKU-213 and KKU-214, using a click chemistry-based enzymatic labeling system, identified using LC-MS/MS, and searched against an OGP database. From the proteomic analysis, a total of 21 OGPs related to cancer progression were identified, of which 12 have not been previously reported. Among these, hnRNP-K, a multifaceted RNA- and DNA-binding protein known as a pre-mRNA-binding protein, was one of the most abundantly expressed, suggesting its involvement in CCA progression. O-GlcNAcylation of hnRNP-K was further verified by anti-OGP/anti-hnRNP-K immunoprecipitations and sWGA pull-down assays. The perpetuation of CCA by hnRNP-K was evaluated using siRNA, which revealed modulation of cyclin D1, XIAP, EMT markers, and MMP2 and MMP7 expression. In native CCA cells, hnRNP-K was primarily localized in the nucleus; however, when O-GlcNAcylation was suppressed, hnRNP-K was retained in the cytoplasm. These data signify an association between nuclear accumulation of hnRNP-K and the migratory capabilities of CCA cells. In human CCA tissues, expression of nuclear hnRNP-K was positively correlated with high O-GlcNAcylation levels, metastatic stage, and shorter survival of CCA patients. This study demonstrates the significance of O-GlcNAcylation on the nuclear translocation of hnRNP-K and its impact on the progression of CCA.

The leading cause of death in cancer patients is metastasis, for which an effective treatment is still necessary. During metastasis, cancer cells aberrantly express several glycans that are correlated with poor patient outcome. This study was aimed toward exploring the effects of O-GlcNAcylation on membranous N-glycans that are associated with the progression of cholangiocarcinoma (CCA). Global O-GlcNAcylation in CCA cells was depleted using specific siRNA against O-GlcNAc transferase (OGT), which transfers GlcNAc to the acceptor proteins. Using an HPLC-Chip/Time-of-Flight (Chip/TOF) MS system, the N-glycans associated with O-GlcNAcylation were identified by comparing the membranous N-glycans of siOGT-treated cells with those of scramble siRNA-treated cells. In parallel, the membranous N-glycans of the parental cells (KKU-213 and KKU-214) were compared with those of the highly metastatic cells (KKU-213L5 and KKU-214L5). Together, these data revealed that high mannose (Hex9HexNAc2) and biantennary complex (Hex5HexNAc4Fuc1NeuAc1) N-linked glycans correlated positively with metastasis. We subsequently demonstrate that suppression of O-GlcNAcylation decreased the expression of these two N-glycans, suggesting that O-GlcNAcylation mediates their levels in CCA. In addition, the ability of highly metastatic cells to migrate and invade was reduced by the presence of Pisum Sativum Agglutinin (PSA), a mannose-specific lectin, further indicating the association of high mannose type N-glycans with CCA metastasis. The molecular mechanism of O-GlcNAc-mediated progression of CCA was shown to proceed via a series of signaling events, involving the activation of Akt/Erk (i), an increase in FOXO3 phosphorylation (ii), which results in the reduction of MAN1A1 expression (iii) and thus the accumulation of Hex9HexNAc2 N-glycans (iv). This study demonstrates for the first time the association between O-GlcNAcylation, high mannose type N-glycans, and the progression of CCA metastasis, suggesting a novel therapeutic target for treatment of metastatic CCA.

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.

Streptococcus gordonii and Streptococcus sanguinis are typically found among the normal oral microbiota but can also cause infective endocarditis. These organisms express cell surface serine-rich repeat adhesins containing "Siglec-like" binding regions (SLBRs) that mediate attachment to α2-3-linked sialic acids on human glycoproteins. Two known receptors for the Siglec-like adhesins are the salivary mucin MG2/MUC7 and platelet GPIbα, and the interaction of streptococci with these targets may contribute to oral colonization and endocarditis, respectively. The SLBRs display a surprising diversity of preferences for defined glycans, ranging from highly selective to broader specificity. In this report, we characterize the glycoproteins in human plasma recognized by four SLBRs that prefer different α2-3 sialoglycan structures. We found that the SLBRs recognize a surprisingly small subset of plasma proteins that are extensively O-glycosylated. The preferred plasma protein ligands for a sialyl-T antigen-selective SLBR are proteoglycan 4 (lubricin) and inter-alpha-trypsin inhibitor heavy chain H4. Conversely, the preferred ligand for a 3'sialyllactosamine-selective SLBR is glycocalicin (the extracellular portion of platelet GPIbα). All four SLBRs recognize C1 inhibitor but detect distinctly different glycoforms of this key regulator of the complement and kallikrein protease cascades. The four plasma ligands have potential roles in thrombosis and inflammation, and each has been cited as a biomarker for one or more vascular or other diseases. The combined results suggest that the interaction of Siglec-like adhesins with different subsets of plasma glycoproteins could have a significant impact on the propensity of streptococci to establish endocardial infections.

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.

Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC-MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host-microbe associations and membrane protein activities.

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.

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.

During the development of recombinant monoclonal antibody (rMAb) drugs, glycosylation receives particular focus because changes in the attached glycans can have a significant impact on the antibody effector functions. The vast heterogeneity of structures that exist across glycosylation sites hinders the in-depth analysis of glycan changes specific to an individual protein within a complex mixture. In this study, we established a sensitive and specific method for monitoring site-specific glycosylation in rMAbs using multiple reaction monitoring (MRM) on an ultrahigh-performance liquid chromatography-triple quadrupole MS (UHPLC-QqQ-MS). Our results showed that irrespective of the IgG subclass expressed in the drugs, the N-glycopeptide profiles are nearly the same but differ in abundances. In all rMAb drugs, a single subclass of IgG comprised over 97% of the total IgG content and showed over 97% N-glycan site occupancy. This study demonstrates the utility of an MRM-based method to rapidly characterize over 130 distinct glycopeptides and determine the extent of site occupancy within minutes. Such multilevel structural characterization is important for the successful development of therapeutic antibodies.

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

Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract.

Photofragment translational spectroscopy was used to study the photodissociation of the methyl perthiyl radical CH3SS at 248 nm. The radical was produced by flash pyrolysis of dimethyl disulfide (CH3SSCH3). Two channels were observed: CH3 + S2 and CH2S + SH. Photofragment translational energy distributions indicate that CH3 + S2 results from C-S bond fission on the ground state surface. The CH2S + SH channel can proceed through isomerization to CH2SSH on the ground state surface but also may involve production of electronically excited CH2S. [ABSTRACT FROM AUTHOR]

Photofragment translational spectroscopy was used to study the photodissociation dynamics of the phenyl radical C6H5 at 248 and 193 nm. At 248 nm, the only dissociation products observed were from H atom loss, attributed primarily to H+o-C6H4 (ortho-benzyne). The observed translational energy distribution was consistent with statistical decay on the ground state surface. At 193 nm, dissociation to H+C6H4 and C4H3+C2H2 was observed. The C6H4 fragment can be either o-C6H4 or l-C6H4 resulting from decyclization of the phenyl ring. The C4H3+C2H2 products dominate over the two H loss channels. Attempts to reproduce the observed branching ratio by assuming ground state dynamics were unsuccessful. However, these calculations assumed that the C4H3 fragment was n-C4H3, and better agreement would be expected if the lower energy i-C4H3+C2H2 channel were included. [ABSTRACT FROM AUTHOR]

The photodissociation dynamics of the tert-butyl radical (t-C4H9) were investigated using photofragment translational spectroscopy. The tert-butyl radical was produced from flash pyrolysis of azo-tert-butane and dissociated at 248 nm. Two distinct channels of approximately equal importance were identified: dissociation to H + 2-methylpropene, and CH3+ dimethylcarbene. Neither the translational energy distributions that describe these two channels nor the product branching ratio are consistent with statistical dissociation on the ground state, and instead favor a mechanism taking place on excited state surfaces. [ABSTRACT FROM AUTHOR]