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Saccharides form one of the major constituents of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response and receptor activation are regulated by glycosylation. In this work, we optimized a capillary electrophoresis method with capacitively coupled contactless conductivity detection for the separation of eight monosaccharides commonly found in glycoproteins, namely D-glucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-fucose, N-acetylneuraminic acid, and D-xylose. A highly alkaline solution of 50 mM sodium hydroxide, 22.5 mM disodium phosphate, and 0.2 mM CTAB (pH 12.4) was used as a background electrolyte in a 10 µm id capillary. To achieve baseline separation of all analytes, a counter-directional pressure of -270 kPa was applied during the separation. The limits of detection of our method were below 7 µg/ml (i.e., 1.5 pg or 1 mg/g protein) and the limits of quantification were below 22 µg/ml (i.e., 5 pg or 3 mg/g protein). As a proof of concept of our methodology, we performed an analysis of monosaccharides released from fetuin glycoprotein by acid hydrolysis. The results show that, when combined with an appropriate pre-concentration technique, the developed method can be used as a monosaccharide profiling tool in glycoproteomics and complement the routinely used LC-MS/MS analysis.

An interplay between receptor-binding properties of influenza viruses (IVs) and spectrum of sialic acid-containing receptors on target cells in birds and mammals determine viral host range, tissue tropism, and pathogenicity. Here, we describe method that allows to characterize binding of IVs to biologically relevant cellular receptors using a conventional solid-phase enzyme-linked assay. In this method, we isolate plasma membranes from respiratory and intestinal epithelial cells of animal origin (Subheading 3.2). We adsorb the membranes in the wells of 96-well ELISA plates, incubate the membrane-coated wells with serially diluted IVs, and determine amounts of IVs attached to the membranes using viral ability to bind peroxidase-labeled sialoglycoprotein fetuin. Based on the concentration dependence of IV binding to the membrane, we estimate binding avidity and number of binding sites. We describe two variants of the assay in Subheadings 3.6 and 3.7 and provide examples.

We describe a method to obtain a comprehensive profile of multiple glycosylations in glycopeptide isoforms. We detected a wide range of abundances of various

Glycosylation is one of the most important post-translational modifications. However, the characterizations of glycopeptides, especially the negatively charged sialoglycopeptides that are associated with various diseases, remain challenging, due to the co-existence with high abundant peptides and the low ionization efficiency of sialoglycopeptides resulting from the carboxyl groups. Therefore, it is essential to develop an efficient enrichment method for sialoglycopeptides. Here, we present a novel derivatization-based enrichment method that can (i) identify linkage isomers of sialic acids by generating mass difference, (ii) unify the net charge of peptides into zero, and (iii) introduce positive charges to sialoglycopeptides by conjugating quaternary ammonium with sialic acid. The derivatization, termed derivatization of sialylated glycopeptides plus (DOSG+), enables efficient enrichment through electrostatic interaction using weak cation exchange (WCX) media. DOSG+ -based WCX enrichment was validated and optimized with samples derived from bovine fetuin. Peptides were removed efficiently (recovery rate1%). The signal intensity of a selected model sialoglycopeptide was increased by ∼30% (suggesting recovery rate100%). The method was employed on human alpha-1 acid glycoprotein (AGP), and recombinant human erythropoietin (EPO), demonstrating the application of DOSG+ -based WCX enrichment on complexed N-linked and O-linked sialoglycopeptides. The method is simple, efficient, and targets small-scale sialoglycopeptide enrichment.

Glycans play an important physiological role and are drawing attention as biomarkers that capture pathophysiological changes. Glycans can be detected by mass spectrometry, but recently matrix-assisted laser desorption/ionization- mass spectrometry imaging (MALDI-MSI) has enabled the visualization of glycans distribution on tissues. In this study, focusing on sialylated glycan (sialoglycans), we investigated the amidation reaction used to visualize glycans distribution, and developed a method of sialic acid derivatization by benzylamidation which is more sensitive than conventional amidation. Furthermore, we adapted this method for visualizing glycans in formalin-fixed paraffin-embedded (FFPE) liver tissue from normal mice and non-alcoholic steatohepatitis (NASH) model mice using MALDI-MSI. As a result, an increase in the distribution of glycan N-Acetylneuraminic acid-(NeuAc) ions was observed in the NASH mouse liver, and the change in glycan structure in the NASH model was clarified.

Dursun, Şükrü ( Aksaray, Yazar ), Background: Freeze-thawing process negatively affects ram spermatozoa in terms of sperm quality, DNA integrity and antioxidant defence system. Thus, antioxidant supplementation of spermatozoa during freeze-thawing is suggested to improve sperm parameters. Objectives: The aim of this study was to determine the effects of fetuin and trehalose added into ram semen extender on sperm parameters, antioxidant parameters, antioxidant-related gene expressions and DNA integrity during the freeze-thawing process, in low glycerol concentration. Methods: Semen samples collected from six mature rams were pooled and splitted into equal aliquots and diluted with a tris-based extender containing different concentrations of glycerol (G5; %5 and G3; %3), fetuin (F; 2.5, 5 and 15 mg/mL) and trehalose (60 mm) as eight groups (G5F0, G5F2.5, G5F5, G5F15, G3F0, G3F2.5, G3F5 and G3F15). Results: G3F5 group resulted in the highest motility, mitochondrial activity and viability and the lowest DNA fragmentation and DNA damage (p < 0.05). Also, G3F0 displayed considerably more cryoprotective effect compared with G5F0 group (p < 0.05) in terms of motility, mitochondrial activity and viability rates. Lipid peroxidation levels decreased in G5F5 group compared with G5F0 group (p < 0.05). The levels of total glutathione increased in G3F2.5 group (p < 0.05) in comparison with the G5F0 group. NQO1 gene levels were upregulated approximately twofold in G5F5, G5F15, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). The levels of GCLC gene were approximately twofold higher in G3F0, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). GSTP1 gene levels were significantly higher with different levels in all treatment groups except for G5F2.5 and G3F0 groups in comparison with G5F0 group (p < 0.05). Conclusions: Co-supplementation of tris-based extender having low glycerol (3%) with trehalose and fetuin to enhance the quality of ram spermatozoa after freeze-thawing process is recommended.

Uromodulin, also known as the Tamm-Horsfall protein or THP, is the most abundant protein excreted in human urine. It is associated with the progression of kidney diseases; therefore, changes in the glycosylation profile of this protein could serve as a potential biomarker for kidney health. The typical glycomics analysis approaches used to quantify uromodulin glycosylation involve time-consuming and tedious glycoprotein isolation and labeling steps, which limit their utility in clinical glycomics assays, where sample throughput is important. Herein, we introduce a radically simplified sample preparation workflow, with direct ESI-MS analysis, enabling the quantification of N-linked glycans that originate from uromodulin. The method omits any glycan labeling steps but includes steps to reduce the salt content of the samples, thereby minimizing ion suppression. The method is effective for quantifying subtle glycosylation differences of uromodulin samples derived from different biological states. As a proof of concept, glycosylation from samples that differ by pregnancy status were shown to be differentiable.

The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods has been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT ™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, and reaction condition length and tag concentration. Optimization of the modified method resulted in 20–100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared to the standard method. Furthermore, the identification of low abundance glycans, such as (Fuc)(1)(Gal)(2)(GlcNAc)(4)(Man)(3)(NeuAc)(1) and (Gal)(3)(GlcNAc)(5)(Man)(3)(NeuAc)(3,) was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC.

Protein glycosylation is involved in many biological processes and physiological functions. Despite the recent advances in LC-MS/MS methodologies, the profiling of site-specific glycosylation is one of the major analytical challenges of glycoprotein analysis. Herein, we first reported the separation of glycopeptide isomers on porous graphitic carbon (PGC)-LC was significantly improved by elevating the separation temperature under basic mobile phases. These findings permitted the isomeric separation of glycopeptides resulting from highly specific enzymatic digestions. The selectivity for different glycan types were studied using bovine fetuin, asialofetuin, IgG, ribonuclease B and alpha-1 acid glycoprotein (AGP) by PGC-LC-MS. Comprehensive structural isomeric separation of glycopeptides was observed by high resolution MS and confirmed by MS/MS. The specific structures of the glycopeptide isomers were identified and confirmed through exoglycosidase digestions. The glycosylation analysis of human AGP revealed the potential use of PGC-LC-MS for extensive glycoprotein analysis for biomarker discovery. This newly developed separation technique was shown as a reproducible and useful analytical method to study site-specific isomeric glycosylation.

Like sialylation, fucose usually locates at the nonreducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans on glycoproteins as well as in their free forms via enzymatic incorporation of fluorophore-conjugated fucose using FUT2, FUT6, FUT7, FUT8 and FUT9. Specifically, we describe the detection of the substrate glycans of these enzymes on fetal bovine fetuin, recombinant H1N1 viral neuraminidase and therapeutic antibodies. The detected glycans include complex and high-mannose N-glycans. By establishing a series of precursors for the synthesis of Lewis X and sialyl Lewis X structures, we not only provide convenient electrophoresis methods for studying glycosylation but also demonstrate the substrate specificities and some kinetic features of these enzymes. Our results support the notion that fucosyltransferases are key targets for regulating the synthesis of Lewis X and sialyl Lewis X structures.

A lectin PCL, from Purpureocillium lilacinum a saprophytic, filamentous fungus was purified from the crude extract of the mycelia using 70% ammonium sulphate precipitation followed by affinity chromatography on mucin-Sepharose 4 B column. PCL is a monomer with an apparent molecular mass of 18.5 kDa as revealed by SDS-PAGE under both reducing and non-reducing conditions. PCL is a blood group non-specific lectin and has highest affinity towards chitin, mucin, asialomucin, fetuin with a MIC of 0.15 µg/mL and also recognizes L-fucose, galactose, lactose, N-acetyl galactosamine, hyaluronic acid. PCL is stable up to 60 °C and within the pH range 4-8. To understand its role in pathogenesis, effect of PCL was evaluated on human corneal epithelial cells (HCECs). PCL showed strong glycan mediated binding to HCECs and PCL showed proinflammatory response at lower concentrations by stimulating secretion of IL-6, 8. In contrast PCL at higher concentrations revealed opposite effect of HCECs growth inhibition. All these results collectively support the involvement of PCL in mediating host pathogen interactions possibly leading to pathogenesis. In addition, considering the entomopathogenic effect of Purpureocillium lilacinum, PCL may be attributed for this beneficiary effect, which needs to be explored.

A new method is presented to decipher information about the isomeric variants of the glycans attached to purified proteins. The method takes advantage of the specificities of glycosidases and lectins, combined with computational integration of the data with information from glycan arrays and mass spectrometry. The application to microspots of proteins makes it compatible with analyzing low-abundance proteins, providing the potential for studying proteins derived from clinical specimens., Graphical Abstract Highlights Method to probe the isomeric variants of the glycans attached to purified proteins.Uses multiple rounds of glycosidase cleavage and lectin profiling.Computation integration of lectin-binding, glycan-array, and mass spectrometry data.Applied to microspots for compatibility with analyzing low-abundance proteins., The difficulty in uncovering detailed information about protein glycosylation stems from the complexity of glycans and the large amount of material needed for the experiments. Here we report a method that gives information on the isomeric variants of glycans in a format compatible with analyzing low-abundance proteins. On-chip glycan modification and probing (on-chip gmap) uses sequential and parallel rounds of exoglycosidase cleavage and lectin profiling of microspots of proteins, together with algorithms that incorporate glycan-array analyses and information from mass spectrometry, when available, to computationally interpret the data. In tests on control proteins with simple or complex glycosylation, on-chip gmap accurately characterized the relative proportions of core types and terminal features of glycans. Subterminal features (monosaccharides and linkages under a terminal monosaccharide) were accurately probed using a rationally designed sequence of lectin and exoglycosidase incubations. The integration of mass information further improved accuracy in each case. An alternative use of on-chip gmap was to complement the mass spectrometry analysis of detached glycans by specifying the isomers that comprise the glycans identified by mass spectrometry. On-chip gmap provides the potential for detailed studies of glycosylation in a format compatible with clinical specimens or other low-abundance sources.

The laser radiation absorbed by cells induces production of reactive oxygen species (ROS), followed by the development of oxidative stress. Proteins are major targets for ROS due to their abundance in biological systems. The aim of the present pilot study was to examine the effects of transcutaneous laser blood irradiation (TLBI), i.e., low-level laser therapy (LLLT) at 830 nm on plasma proteome in Wistar rats. Rats were irradiated in the heart area (i.e. coronary arteries) daily (i.e., for 9-day period), by commercially available GaAsAl diode laser (Maestro/CCM, Medicom Prague, Czech Republic, λ=830 nm, power density 450mW/cm2, daily dose 60,3 J/ cm(2), irradiation time 134 sec). The comparison of blood plasma proteome from irradiated and non-irradiated rats was performed utilizing 2D electrophoresis followed by MALDI TOF/TOF mass spectrometry. LLLT led to a quantitative change in the acute phase proteins with antioxidant protection i.e., haptoglobin (log2 fold change (FC)=3.5), hemopexin (log2 FC=0.5), fibrinogen gamma (log2 FC=1.4), alpha-1-antitrypsin (log2 FC=-2.2), fetuin A (log2 FC=-0.6) and fetuin B (log2 FC=-2.3). In comparison to conventional biochemical methods, the changes in protein levels in blood plasma induced by LLLT offer a deeper insight into the oxidative stress response.

Paratuberculosis (PTB) is a chronic contagious granulomatous enteritis of wild and domestic ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB causes considerable economic losses to the dairy industry through decreased milk production and premature culling. PTB-affected cattle undergo a subclinical stage without clinical signs and initiate fecal shedding of MAP into the environment. Current diagnostic tools have low sensitivity for the detection of subclinical PTB infection. Therefore, alternative diagnostic tools are required to improve the diagnostic sensitivity of subclinical PTB infection. In this study, we performed ELISA for three previously identified host biomarkers (fetuin, alpha-1-acid glycoprotein, and apolipoprotein) and analyzed their diagnostic performance with conventional PTB diagnostic methods. We observed that serum fetuin levels were significantly lowered in the subclinical shedder and clinical shedder groups than in the healthy control group, indicating its potential utility as a diagnostic biomarker for bovine PTB. Also, fetuin showed an excellent discriminatory power with an AUC = 0.949, a sensitivity of 92.6%, and a specificity of 94.4% for the detection of subclinical MAP infection. In conclusion, our results demonstrated that fetuin could be used as a diagnostic biomarker for enhancing the diagnostic sensitivity for the detection of subclinical MAP infections that are difficult to detect based on current diagnostic methods.

We used an electron-transfer carbohydrate-mimetic peptide (YYYYC) to construct an electrochemical cytosensing system. Magnetic beads were modified with either asialofetuin (ASF) or soybean agglutinin (SBA) to evaluate the effect on cell sensing. Because SBA binds to the galactose residue that exists at the terminals of the carbohydrate chains in ASF, the target protein was accumulated on the protein magnetic beads. SBA is an example of N-acetylgalactosamine- and galactose-binding proteins that readily combine with YYYYC. When the peptides and protein-immobilized beads competed for a target protein, the peak current of the peptides changed according to the concentration of the protein at the 10−12 M level. Next, human myeloid leukemia cells (K562 cell) were measured using the peptide and the carbohydrate chains on the cell surface that recognize SBA. The electrode response was linear to the number of K562 cells and ranged from 1.0 × 102 to 5.0 × 103 cells mL−1. In addition, detection of a human liver cancer cell (HepG2 cell) was carried out using interactions with the peptide, the ASF receptors in HepG2 cells, and the carbohydrate chains of ASF. The peak currents were proportional and ranged between 5.0 × 101 and 1.5 × 103 cells mL−1. When the values estimated from an electrochemical process were compared with those obtained by ELISA, the results were within the acceptable range of measurement error.

Polysialic acids are bioactive carbohydrates found in eukaryotes and some bacterial pathogens. The bacterial polysialyltransferases (PSTs), which catalyze the synthesis of polysialic acid capsules, have previously been identified in select strains of Escherichia coli and Neisseria meningitidis and are classified in the Carbohydrate-Active enZYmes Database as glycosyltransferase family GT-38. In this study using DNA sequence analysis and functional characterization we have identified a novel polysialyltransferase from the bovine/ovine pathogen Mannheimia haemolytica A2 (PSTMh). The enzyme was expressed in recombinant form as a soluble maltose-binding-protein fusion in parallel with the related PSTs from E. coli K1 and N. meningitidis group B in order to perform a side-by-side comparison. Biochemical properties including solubility, acceptor preference, reaction pH optima, thermostability, kinetics, and product chain length for the enzymes were compared using a synthetic fluorescent acceptor molecule. PSTMh exhibited biochemical properties that make it an attractive candidate for chemi-enzymatic synthesis applications of polysialic acid. The activity of PSTMh was examined on a model glycoprotein and the surface of a neuroprogenitor cell line where the results supported its development for use in applications to therapeutic protein modification and cell surface glycan remodelling to enable cell migration at implantation sites to promote wound healing. The three PSTs examined here demonstrated different properties that would each be useful to therapeutic applications. © 2013 Lindhout et al.

Despite tremendous complexity in glycan structure, sialic acid (SA) provides an analytically accessible index for glycosylation, owing to its uniquely anionic nature and glycan-chain terminal occupation. Taking advantage of boronic acid (BA) based SA-recognition chemistry, we here demonstrate a label-free, no enzymatic, potentiometric determination of fetuin, a blood-circulating glycoprotein implicated in physiological and various pathological states. A phenylboronic acid (PBA) ω-end-functionalized poly(ethylene glycol) (PEG) with an α-tethering unit bearing pendent alkyne groups was "grafted-to" a gold electrode modified with 11-azide-undecathiol by a copper-catalyzed azide-alkyne cycloaddition reaction. Using the electrode, fetuin was potentiometrically detectable with a μM-order-sensitivity that is comparable to what is found in blood-collected specimen. Our finding may have implications for developing a remarkably economic hemodiagnostic technology with ease of downsizing and mass production.

Fetuin is an abundant blood protein that participates in multiple biological processes, including the transport and regulation of calcium. Fetuin is also known to have a high affinity for uranium (as the uranyl dioxo cation) and plutonium, thus it has been suggested as one of the main endogenous chelating biomolecules involved in the transport of actinides following an internal uptake event. Nevertheless, no direct measurements of its affinity for f-elements beside these two actinides have been reported. Here, we investigate the interaction between fetuin and trivalent lanthanides, such as samarium, europium, terbium, and dysprosium, by mass spectrometry and fluorescence spectroscopy. Mass spectrometry results indicated that fetuin has four metal binding sites for the metal ions studied. Upon formation, the metal-protein complexes showed luminescence emission as a result of antenna sensitization of the metal ions, whose photophysics were characterized and exploited to perform direct spectrofluorimetric titrations. Furthermore, the thermodynamic constants were calculated for all complexes, confirming the formation of stable complexes with log [Formula: see text] values between 26 and 27. In characterizing the affinity of the serum protein fetuin for several f-elements, this study expands upon the initial findings focused on uranyl and plutonium, and contributes to a better understanding of the internal distribution and deposition of lanthanides, potentially representative of trivalent actinides.

O-glycosylation is a highly diverse and complex form of protein post-translational modification. Mucin-type O-glycosylation is initiated by the transfer of N-acetyl-galactosamine (GalNAc) to the hydroxyl group of serine, threonine and tyrosine residues through catalysis by a family of glycosyltransferases, the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (E.C. 2.4.1.41) that are conserved across metazoans. In the last decade, structural characterization of glycosylation has substantially advanced due to the development of analytical methods and advances in mass spectrometry. However, O-glycosite mapping remains challenging since mucin-type O-glycans are densely packed, often protecting proteins from cleavage by proteases. Adding to the complexity is the fact that a given glycosite can be modified by different glycans, resulting in an array of glycoforms rising from one glycosite. In this study, we investigated conditions of solid phase extraction (SPE) enrichment, protease digestion, and Electron-transfer/Higher Energy Collision Dissociation (EThcD) fragmentation to optimize identification of O-glycosites in densely glycosylated proteins. Our results revealed that anion-exchange stationary phase is sufficient for glycopeptide enrichment; however, the use of a hydrophobic-containing sorbent was detrimental to the binding of polar-hydrophilic glycopeptides. Different proteases can be employed for enhancing coverage of O-glycosites, while derivatization of negatively charged amino acids or sialic acids would enhance the identification of a short O-glycopeptides. Using a longer than normal electron transfer dissociation (ETD) reaction time, we obtained enhanced coverage of peptide bonds that facilitated the localization of O-glycosites. O-glycosite mapping strategy via proteases, cut-off filtration and solid-phase chemoenzymatic processing. Glycopeptides are enriched by SPE column, followed by release of N-glycans, collection of higher MW O-glycopeptides via MW cut-off filter, O-glycopeptide release via O-protease, and finally detected by LC-MS/MS using EThcD.

Precise and automated analysis of site-specific

Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of α2,3/α2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy. After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of α2,3-sailoglycopeptides then generate unique reporters to distinctly differentiate those of α2,6-sailoglycopeptide isomers. With this strategy, a total of 1236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.

Fetuin is an abundant blood protein that participates in multiple biological processes, including the transport and regulation of calcium. Fetuin is also known to have a high affinity for uranium (as the uranyl dioxo cation) and plutonium, thus it has been suggested as one of the main endogenous chelating biomolecules involved in the transport of actinides following an internal uptake event. Nevertheless, no direct measurements of its affinity for f-elements beside these two actinides have been reported. Here, we investigate the interaction between fetuin and trivalent lanthanides, such as samarium, europium, terbium, and dysprosium, by mass spectrometry and fluorescence spectroscopy. Mass spectrometry results indicated that fetuin has four metal binding sites for the metal ions studied. Upon formation, the metal-protein complexes showed luminescence emission as a result of antenna sensitization of the metal ions, whose photophysics were characterized and exploited to perform direct spectrofluorimetric titrations. Furthermore, the thermodynamic constants were calculated for all complexes, confirming the formation of stable complexes with log [Formula: see text] values between 26 and 27. In characterizing the affinity of the serum protein fetuin for several f-elements, this study expands upon the initial findings focused on uranyl and plutonium, and contributes to a better understanding of the internal distribution and deposition of lanthanides, potentially representative of trivalent actinides.

The neuraminidase (NA) of influenza A viruses (IAV) is a structurally and antigenically important envelope glycoprotein. There are eleven known subtypes of NA of which two, N1 and N2, circulate in swine. The sialidase activity of NA is required for the release of nascent virus particles from infected cell membranes and inhibition of NA enzymatic activity can significantly reduce virus titers and duration of infection. Efforts to improve IAV vaccine technology in humans have focused on the generation of neuraminidase inhibiting (NAI) antibodies and should be considered in swine as well. The enzyme-linked lectin assay (ELLA) conducted in 96-well plates has enabled high-throughput analysis of serum samples for NAI antibody titers. Through the use of reverse genetics, custom antigen panels and antisera can be generated to encompass the antigenically diverse population of NA that circulate in swine. The ELLA is a robust method to assess NAI antibody titers and characterize the antigenic difference between NA antigens.

Sialylation, an important form of glycosylation, is involved in many biological processes and plays an important role in the development of diseases. However, due to the low abundance among various glycosylation and lack of efficient enrichment method with high specificity, the study of sialylation remains a challenge. Herein, multi-histidine modified microspheres (MHM) were synthesized to enrich sialylated glycopeptides. It was found that MHM could selectively enrich sialylated glycopeptides from over 100 times of non-sialylated glycopeptides, which indicated MHM possessed good enrichment specificity towards sialylated glycopeptides. Furthermore, MHM were utilized to the large-scale analysis of protein sialylation, and 510 intact glycopeptides were identified with over 94.5% sialylated glycopeptide specificity from 4 μL human serum. The good specificity could be attributed to the synergistic effect by the electrostatic interaction and hydrophilic interaction. Hence, MHM could provide an alternative approach for the analysis of site-specific sialylation at proteome level from complex biological samples.

INTRODUCTION Insulin resistance may develop with Type 1 diabetes. Insulin resistance is currently recognized by the estimated glucose disposal rate. Serum fetuin has been accused as a risk factor for metabolic syndrome. AIM To determine the relationship between the serum fetuin and insulin resistance in Type 1 diabetes subjects and the effect of short-term Metformin therapy on this relationship. METHODS 40 T1DM male ≥ 18 years of age were screened for insulin resistance (defined using estimated glucose disposal rate). 20 subjects (Group I) were insulin resistant with a mean estimated glucose disposal rate of (7.15±0.37 mg/kg/min) while 20 subjects (Group II) were non-insulin resistant with a mean estimated glucose disposal rate of (9.08±0.42 mg/kg/min). Fasting blood sugar, 2 hours-post prandial blood sugar, HbA1c%, C-peptide, lipid profile, highly sensitive-C reactive protein, and serum fetuin were assessed. Group I were given 1gm Metformin twice daily for 3 months as an add-on to their insulin regimen. All anthropometric and laboratory parameters were reassessed at the end of the 3 months. RESULTS Group I had a higher age, BMI and waist/hip ratio, FBS, PPBS, HbA1c%, TC, LDL-C, TG, Hs-CRP and serum fetuin (ρ ≤ 0.001), and a lower C-peptide (ρ=0.001). Fetuin showed a positive correlation with age, FBS, HbA1c%, and Hs-CRP. After Metformin therapy, FBS, PPB and HbA1c%, Hs- CRP and fetuin decreased (ρ≤0.001) while eGDR and insulin dose in units/kg increased (ρ

Glycosylation analysis of proteins from biological sources utilizing mass spectrometry based approaches is challenging due to the relatively low abundance of glycopeptides, the structural diversity of glycans, and the coexisting matrices. In this study, a customized dextran-bonded silica-based stationary phase was introduced for selective enrichment of glycopeptides and glycans from complex biological samples. This material has exhibited superior selectivity and broader glycosylation site coverage over commercial Sepharose in glycoproteomic evaluation. Additionally, the glycomic analysis of fetuin, α1 -acid glycoprotein, and human serum N-glycome also indicated the relatively higher sensitivity, selectivity, and glycoform coverage of dextran-bonded silica than that of Sepharose and porous graphitized carbon. Therefore, the dextran-bonded silica is expected to make contributions in the fields of glycoproteomics and glycomics.

Lectin inspired polymers were prepared through modification of silica microspheres with Ser-Asp (SD). This functional polymer showed distinct adsorption and retention towards different disaccharides and demonstrated high-efficiency enrichment of glycopeptides.

To date, few tools are available for the analysis of the glycome without derivatization, a process which is known to introduce issues such as differential loss of sialic acid and incomplete labeling. We have previously reported the use of ion chromatography-mass spectrometry (IC-MS) to analyze native sialylated and sulfated glycans. Here, we introduce improvements to IC column technology, enabling the separation of neutral glycans while maintaining charge separation capabilities. When implemented in an IC-MS workflow, this enables the structural characterization of a broad array of chemically distinct glycans. With the newly developed IC column and modified IC-MS instrumentation configuration, we qualitatively investigated O-glycome profiles in bovine fetuin and porcine gastric mucins. The improved chromatographic resolution in combination with high-resolution MS data present a powerful tool for glycan structural identification.

Fetuin is a biocompatible plasma protein and strongly enhances phagocytosis of bacteria, DNA and apoptotic cells by peripheral blood cells such as monocytes, macrophages and dendritic cells. We developed a novel gene delivery system: ternary complexes constructed with pDNA, polyethylenimine (PEI) and fetuin. Without covalent binding, fetuin was able to coat pDNA-PEI complexes, and stable anionic nanoparticles formed at a weight ratio greater than 30. Optimised pDNA-PEI-fetuin complexes significantly decreased the cytotoxicity of pDNA-PEI complexes in the melanoma cell line B16F10. Furthermore, the pDNA-PEI-fetuin complexes had higher transgene efficiency compared to that of commercial lipofectin previously reported in B16F10 cells despite an anionic surface. The pDNA-PEI-fetuin complexes did not agglutinate with erythrocytes. The pDNA-PEI-fetuin complexes had high gene expression in the spleen after intravenous administration in mice. Thus, the pDNA-PEI-fetuin complexes were a useful in vivo gene delivery system with tropism for the spleen.

To overcome the challenges in the analysis of protein glycosylation, we have developed a comprehensive and universal tool through permethylation of glycopeptides and their tandem mass spectrometric analysis. This method has the potential to simplify glycoprotein analysis by integrating glycan sequencing and glycopeptide analysis in a single experiment. Moreover, glycans with unique glycosidic linkages, particularly from prokaryotes, which are resistant to enzymatic or chemical release, could also be detected and analyzed by this methodology. Here we present a strategy for the permethylation of intact glycopeptides, obtained via controlled protease digest, and their characterization by using advanced mass spectrometry. We used bovine RNase B, human transferrin, and bovine fetuin as models to demonstrate the feasibility of the method. Remarkably, the glycan patterns, glycosylation site, and their occupancy by N-glycans are all detected and identified in a single experimental procedure. Acquisition on a high resolution tandem-MSn system with fragmentation methodologies such as high-energy collision dissociation (HCD) and collision induced dissociation (CID), provided the complete sequence of the glycan structures attached to the peptides. The behavior of 20 natural amino acids under the basic permethylation conditions was probed by permethylating a library of short synthetic peptides. Our studies indicate that the permethylation imparts simple, limited, and predictable chemical transformations on peptides and do not interfere with the interpretation of MS/MS data. In addition to this, permethylated O-glycans in unreduced form (released by β elimination) were also detected, allowing us to profile O-linked glycan structures simultaneously.

Mucin-type O-glycans (O-glycans) are one of the most common glycans attached to proteins. To develop an optimized glycomic analysis protocol, O-glycans were released from glycoproteins using hydrazine, ammonia, or sodium hydroxide treatment, followed by hydrophilic interaction liquid chromatography to evaluate O-glycan release. We found that porcine gastric mucin or bovine fetuin treated at 60 °C for 6 h with hydrazine gas in the presence of malonic acid yielded O-glycans with only a small amount of degraded, so-called "peeled" products. Ammonia treatment also yielded intact O-glycans but with additional peeled products containing GlcNAc at the reducing end. In contrast, sodium hydroxide treatment yielded mainly peeled glycans, including those containing GlcNAc at the reducing end. Importantly, O-glycans obtained from rat gastric mucin treated with hydrazine and labeled with anthranilic acid had a nearly identical profile following hydrophilic interaction liquid chromatography as permethylated O-glycan alditols analyzed by mass spectroscopy. Taken together, the data suggest that glycan release using hydrazine treatment, followed by high-performance liquid chromatography after fluorescent labeling, is a suitable method for glycomic analysis of mucin-type O-glycans.

Combining powerful selectivity, high stability, convenient operation, mild condition, and eco-friendliness, a novel graphitic carbon nitride (g-C3N4)-based enrichment method of intact sialoglycopeptides (SGs) was developed. The intact SGs could be simply enriched and separated from protein tryptic digests by hydrogen bonding without damage of glycan structures due to the specific structure of g-C3N4. By optimizing the enrichment and elution conditions, 45 and 38 SGs were detected from the tryptic digests of bovine fetuin and transferrin, respectively. Under the synergistic effect of hydrogen bonding and electrostatic adsorption, the SGs could be enriched simply in less than 2 h with a detection limit of 50 fmol. The method is repeatable due to the high stability of g-C3N4 and the simple protocol of the method, indicating the potential application of g-C3N4 in efficient and selective enrichment of intact SGs.

Insufficient sialylation can result in rapid clearance of therapeutic glycoproteins by intracellular degradation, which is mainly mediated by asialoglycoprotein receptors (ASGPRs) on hepatic cells. In contrast, for glycoproteins, a long half-life is often related to high level of terminal sialic acid. These could be extremely important for insufficient sialylated biomedicines in clinic, and development of therapeutic glycoproteins in laboratory. However, how the desialylated glycoproteins are removed and how to evaluate the ASGPRs mediated endocytosis in vitro needs further investigate. Herein we described an integrative characterization of ASGPRs in vitro to elucidate its endocytosis properties. The endocytosis was determined by a fluorescence-based quantization method. The results showed that the ASGPRs could bind to poorly sialylated glycoproteins including asialofetuin and low sialylated recombinant Factor VIIa with a relatively higher ASGPRs binding affinity, and induce a more rapid endocytosis in vitro. Moreover, the mechanism under the internalization of ASGPRs was also investigated, which was found to depend on clathrin and caveolin. Utilizing the relative fluorescence quantification can be suitable for measurement of insufficient sialylated glycoprotein endocytosis and quality control of therapeutic glycoproteins, which could be useful for the understanding of the development of therapeutic glycoproteins.

We have recently generated a highly efficient 1,2-α-l-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-α-l-fucosidase from Bifidobacterium bifidum JCM 1254. This synthase could specifically introduce H-antigens (Fucα1-2Gal) into the non-reducing ends of oligosaccharides and in O-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-α-l-fucosynthase by demonstrating its ability to insert Fuc residues into N- and O-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics.

The diversity of O-linked glycan structures has drawn increasing attention due to its vital biological roles. However, intact O-glycopeptides with different glycans are typically not well elucidated using the current methods. In this work, an integrated strategy was developed for comprehensive analysis of O-GalNAc glycosylation by combining hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type collision induced decomposition (beam-CID) detection, and in silico deglycosylation method for spectra interpretation. In this strategy, the intact O-GalNAc glycopeptides were selectively enriched and the original spectra obtained by time-of-flight (TOF)-CID were preprocessed using an in silico deglycosylation method, enabling direct searching without setting multiple glycosylation modifications, which could significantly decrease the search space. This strategy was applied to analyze the O-GalNAc glycoproteome of human serum, leading to identification of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About 81% of the glycopeptides contained at least one sialic acid, which could reveal the microheterogeneity of O-GalNAc glycosylation. Up until now, this is the largest data set of intact O-GalNAc glycoforms from complex biological samples at the proteome level. Furthermore, this method is readily applicable to study O-glycoform heterogeneity in other complex biological systems.

Peptide-N-glycosidase F (PNGase F) is the most frequently used enzyme to release N-glycan from glycoproteins in glycomics; however, the releasing process using PNGase F is tedious and can range in duration from hours to overnight. Recently, efforts have been made to accelerate this enzymatic reaction, and they include the use of microwave irradiation, ultrahigh pressure, enzyme immobilization, and other techniques. Here, we developed a novel method combining the oriented immobilization of PNGase F on magnetic particles and microwave-assisted enzymatic digestion techniques to achieve highly efficient release of N-glycans. The oriented immobilization of PNGase F on magnetic particles utilizes the affinity of its co-expressed His-tag towards iminodiacetic acid-Nickel modified magnetic particles. Compared with non-oriented immobilization, the oriented immobilization of PNGase F exhibits several advantages including tolerance to high temperature (52 °C) and the ability to retain strong activity after more than five reuses. When used in combination with microwave irradiation, efficient N-glycan removal from ribonuclease B was achieved within 5 min. The proposed strategy was also used to release glycan from fetuin and human serum and has proven to provide a promising deglycosylation method for the characterization of protein glycosylation.

The study of protein glycosylation is important to deepening the current understanding of its biological functions as well as to elucidate novel biomarkers of glycosylation. However, glycopeptides must be enriched prior to analysis due to their naturally low abundance. In this study, tryptophan functionalized polymer materials (denoted as Poly-Trp) were synthesized to serve as a novel biomimetic polymers. The resulting materials were characterized by various methods including scanning electron microscopy, thermal analysis, and infrared spectrometry, validating the successful synthesis of Poly-Trp. The retention of glycopeptides on Poly-Trp was investigated revealing that the retention ability decreased as the acetonitrile content of the mobile phase decreased and its acidity increased. Poly-Trp exhibited higher enrichment selectivity for glycopeptides from bovine fetuin than the commercial material ZIC-HILIC, and aminated silica which could defect the interference of 100-fold amount of substance ratios of bovine serum albumin. Poly-Trp is expected to have further applications in the purification of biological samples for the study of glycosylation.

Sialic acids, terminal structures of sialylated glycoconjugates, are widely distributed in animal tissues and are often involved in intercellular recognitions, including some bacteria and viruses. Mycoplasma mobile, a fish pathogenic bacterium, binds to sialyloligosaccharide (SO) through adhesin Gli349 and glides on host cell surfaces. The amino acid sequence of Gli349 shows no similarity to known SO-binding proteins. In the present study, we predicted the binding part of Gli349, produced it in Escherichia coli and proved its binding activity to SOs of fetuin using atomic force microscopy. Binding was detected with a frequency of 10.3% under retraction speed of 400 nm/s and was shown to be specific for SO, as binding events were competitively inhibited by the addition of free 3′-sialyllactose. The histogram of the unbinding forces showed 24 pN and additional peaks. These results suggested that the distal end of Gli349 constitutes a novel sialoadhesin domain and is directly involved in the gliding mechanism of M. mobile., This study identified binding activity to sialyloligosaccharide in the foot domain of Gli349, a large 349 kDa molecular weight protein that works as a leg in Mycoplasma mobile gliding.

The chicken β-galactoside α2,3-sialyltransferase 1, 2, and 5 (ST3Gal1, 2, and 5) genes were cloned, and their enzymes were expressed in 293FT cells. ST3Gal1 and 2 exhibited enzymatic activities toward galactose-β1,3-N-acetylgalactosamine and galactose-β1,3-N-acetylglucosamine. ST3Gal5 only exhibited activity toward lactosylceramide. ST3Gal1 and 2 and previously cloned ST3Gal3 and 6 transferred CMP-sialic acid to asialofetuin. Reverse-transcription-quantitative PCR indicated that ST3Gal1 was expressed at higher levels in the trachea, lung, spleen, and magnum, and the strong expression of ST3Gal5 was observed in the spleen, magnum, and small and large intestines. ST3Gal1, 5, and 6 were also expressed in the tubular gland cells of the magnum, which secretes egg-white proteins. ST3Gal1, 5, and 6 were expressed in the egg chorioallantoic membrane, in which influenza viruses are propagated for the production of vaccines.

N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.

The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in α2-3 and α2-6 linkages, while these glycans remain attached to peptides.

Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.

Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis and its detoxified form is one of the major protective antigens in vaccines against whooping cough. Ideally, PTx in the vaccine should be completely detoxified while still preserving immunogenicity. However, this may not always be the case. Due to multilevel reaction mechanisms of chemical detoxification that act on different molecular sites and with different production processes, it is difficult to define a molecular characteristic of a pertussis toxoid. PTx has two functional distinctive domains: the ADP-ribosyltransferase enzymatic subunit S1 (A-protomer) and the host cell binding carbohydrate-binding subunits S2-5 (B-oligomer); and in this study, we investigated the effect of different detoxification processes on these two functional activities of the residual PTx in toxoids and vaccines currently marketed worldwide using a recently developed in vitro biochemical assay system. The patho-physiological activities in these samples were also estimated using the in vivo official histamine sensitisation tests. Different types of vaccines, detoxified by formaldehyde, glutaraldehyde or by both, have different residual functional and individual baseline activities. Of the vaccines tested, PT toxoid detoxified by formaldehyde had the lowest residual PTx ADP-ribosyltransferase activity. The carbohydrate binding results detected by anti-PTx polyclonal (pAb) and anti-PTx subunits monoclonal antibodies (mAb) showed specific binding profiles for toxoids and vaccines produced from different detoxification methods. In addition, we also demonstrated that using pAb or mAb S2/3 as detection antibodies would give a better differential difference between these vaccine lots than using mAbs S1 or S4. In summary, we showed for the first time that by measuring the activities of the two functional domains of PTx, we could characterise pertussis toxoids prepared from different chemical detoxification methods and this study also highlights the potential use of this in vitro biochemical assay system for in-process control.

Aims Clinical studies have shown that very low protein diet (VLPD) has negative effects on long-term survival. It remains unclear why VLPD induces premature death. The present study determined the underlying mechanism whereby VLPD exerts its harmful effects on uremic rats. Main methods Rats were divided into four groups and fed a normal diet or diets containing 0.3% adenine and low/normal protein with high/low phosphate. After 6 weeks, body weight, urinary biochemistry (creatinine and phosphate), serum biochemical parameters (urea, creatinine, fibroblast growth factor 23, albumin, and fetuin-A), systemic inflammatory markers (serum tumor necrosis factor-alpha and urinary 8-hydroxy-2′-deoxyguanosine), calcium content in the aorta, and serum calcium-phosphate precipitates were evaluated. Hepatic mRNA levels were also determined. Key findings Rats fed the diet containing 0.3% adenine developed severe azotemia. Rats fed VLPD developed malnutrition (decreases in body weight, serum albumin and fetuin-A levels, and urinary creatinine excretion) and systemic inflammation (increases in serum tumor necrosis factor-α and urinary 8-hydroxy-2′-deoxyguanosine) independent of phosphate status. VLPD decreased the serum fetuin-A level and hepatic fetuin-A synthesis and increased serum calcium-phosphate precipitates, a marker of calciprotein particle. A high-phosphate diet induced arterial medial calcification, which was enhanced by VLPD. Serum calcium-phosphate precipitate levels were correlated with the degree of inflammation, malnutrition, and aortic calcium content. Dietary phosphate restriction prevented VLPD-enhanced vascular calcification, but could not halt inflammation and malnutrition induced by VLPD. Significance VLPD enhances inflammation, malnutrition, and vascular calcification in uremic rats, among which only vascular calcification is prevented by dietary phosphate restriction.

Tumor genesis is accompanied by glycosylation of related proteins. Glycoprotein is usually regarded as a tumor marker since glycoproteins are consumed remarkably more by the cancer cells than the normal ones. In this paper, the terahertz time-domain attenuated total reflection (ATR) technique is applied to inspect the glycoprotein solution from a concentration gradient of 0.2 mg/ml to 50 mg/ml. A significant nonlinear relationship between the absorption coefficient and the concentrations has been discovered. The influence of the dynamical hydration shell around glycoprotein molecules on the absorption coefficient is discussed and the phenomenon is explained by the concepts of THz excess and THz defect. In order to identify glycoproteins, features are obtained by composite multiscale entropy (CMSE) method and clustered by the K-means algorithm. The results indicate that features extracted by the CMSE method are better than the Principal Component Analysis (PCA) method in both specificity and sensitivity of recognition. Meanwhile, the absorption coefficient and dielectric loss angle tangent are more suitable for qualitative identification. Research shows that the CMSE method has important directive significance for analyzing glycoprotein terahertz spectroscopy. And it has the potential for glycoprotein related tumor markers identification using terahertz technology in medical applications.

A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry (named SiO2-DVS-GSH). The successful synthesis of the SiO2-DVS-GSH material was confirmed by FT-IR, elemental analysis, and zeta potential analysis. The effects of water content, pH value, and salt concentration in the mobile phase on the model compound (uracil, uridine, cytosine, cytidine, guanosine, xanthosine, orotic acid) retention was studied, and a hydrophilic interaction liquid chromatography (HILIC) retention feature together with electrostatic interaction of the SiO2-DVS-GSH material was observed. The prepared stationary phase was further applied for the separation of oligosaccharide. In addition, the SiO2-DVS-GSH material displayed remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment from bovine fetuin tryptic digests, even at a mass ratio of 1:1000 (w/w) to bovine serum albumin (BSA, non-glycosylated protein), showing superior performance compared to commercial ZIC-HILIC material. Moreover, the SiO2-DVS-GSH material behaved well in the N-glycopeptides' enrichment from human serum, demonstrating its promising potential for glycoproteomics of complex biological samples. Graphical Abstract A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry, and it shows remarkable selectivity and specificity for the sialylated N-glycopeptides' enrichment.

Described in several epithelial cancer cells, Tn- (GalNAcα1-O-Ser/Thr) and T- (Galβ3GalNAcα1-O-Ser/Thr) antigens are examples of tumor-associated antigens. Increased expression of Tn- and T-antigens is associated with tumor invasion and metastasis, and patients with high concentration of anti-Tn and anti-T antibodies have a more benign evolution of pathology. Asialofetuin (ASF) and ovine submaxillary mucin (OSM) are two glycoproteins that expose T- and Tn-antigen, respectively. In this work, using ASF or OSM we affinity-purified anti-T and anti-Tn antibodies from normal human plasma and tested their ability to specifically recognize tumor human tissues. Whereas purified anti-T antibodies (purity degree increase of 127-fold, and 22% recovery) were mainly IgG, for purified anti-Tn antibodies (purity degree enhancement of 125-fold, and 26% yield) the IgM fraction was predominant over the IgG one. IgG2 subclass was significantly enriched in both purified antibody samples. Purified antibodies did not bind normal human tissue (0/42), although recognized malignant tissues from different origin such as colon carcinoma (11/77 by anti-Tn; 7/79 by anti-T), breast carcinoma (10/23 by anti-Tn; 7/23 by anti-T), and kidney carcinoma (45/51 by anti-Tn; 42/51 by anti-T). Our results suggest that purified human anti-Tn and anti-T antibodies have a potential as anti-tumor therapeutic agents; restoring their levels in human sera could positively affect the evolution of patients with epithelial tumor pathologies. Fil: Zlocowski, Natacha. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Grupe, Verónica Maria. Fundación Para El Progreso de la Medicina; Argentina Fil: Garay, Yohana Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Nores, Gustavo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Lardone, Ricardo Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Irazoqui, Fernando Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

Rationale: Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed‐phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site‐specific glycosylation. Methods: Digests of protein standards (horse myoglobin, bovine fetuin and β‐casein) were analyzed with a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) system by coupling an Agilent 1100 HPLC system to a Synapt G2‐Si HDMS (Waters). Peptides were separated using a HyperCarb PGC column (300 μm i.d. × 100 mm) packed with 3 μm particles. MS/MS data were collected in data‐dependent mode and three MS/MS scans were acquired after the full MS scan. RAW data were transformed to .mgf by PLGS (Waters) and searched against the Swissprot database by Mascot. Chromatograms and MS/MS spectra of identified compounds were extracted with Masslynx (Waters) and imported to Origin for analysis. Glycan composition and peptide sequence were manually annotated. Results: PGC/MS/MS enabled accurate quantitation of the stoichiometry of specific phosphorylation sites from β‐casein by efficient separation of the phosphopeptide and its non‐phosphorylated counterpart, which cannot be achieved by reversed‐phase chromatography. PGC/MS/MS also enabled comprehensive characterization of protein sialoglycosylation as isomeric glycopeptides with different combinations of α2‐3‐ and α2‐6‐linked sialic acids can be separated and the ratios of each combination were verified by exoglycosidase digestion. Conclusions: PGC has demonstrated superior separation of peptides with phosphorylation and glycosylation and can be used as an alternative in the proteomic characterization of post‐translational modifications (PTMs) by polar groups.

Analysis of glycans via a porous graphitized carbon liquid chromatography (PGC-LC) coupled with electrospray ionization (tandem) mass spectrometry (ESI-MS(/MS)) is a powerful analytical method in the field of glycomics. Isobaric glycan structures can be identified reliably with the help of PGC-LC separation and subsequent identification by ESI-MS(/MS) in negative ion mode. In an effort to adapt PGC-LC-ESI-MS(/MS) to the nano-scale operation, spray instability along the nano-PGC-LC gradient was repeatedly observed on an LTQ Orbitrap Elite mass spectrometer equipped with a standard nano-electrospray ionization source. A stable electrospray was achieved with the implementation of a post-column make-up flow (PCMF). Thereby, acetonitrile was used to supplement the eluate from the nano-PGC-LC column. The improved spray stability enhanced detection and resolution of glycans during the analysis. This was in particular the case for smaller O-glycans which elute early in the high aqueous content regime of the nano-PGC-LC elution gradient. This study introduces PCMF as an easy-to-use instrumental adaptation to significantly improve spray stability in negative ion mode nano-PGC-LC-ESI-MS(/MS)-based analysis of glycans.