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Glycopeptides
Kinds of Glycopeptides Terms modified by Glycopeptides Selected AbstractsGlycopeptide and glycoprotein synthesis involving unprotected carbohydrate building blocks,MEDICINAL RESEARCH REVIEWS, Issue 6 2005Zhongwu Guo Abstract This review summarizes the chemical and chemoenzymatic synthesis of glycopeptides and glycoproteins using unprotected carbohydrates as key intermediates. The synthetic methods covered herein include the convergent synthesis of glycopeptides by chemoselective ligation of peptides and free glycans, solution- and solid-phase synthesis of glycopeptides by sequential peptide elongation with unprotected glycosyl amino acids or short glycopeptides as building blocks, and the synthesis of glycopeptides by enzymatic and/or chemical elongation of the free glycans. The use of unprotected carbohydrates in these syntheses can circumvent the final-stage carbohydrate deprotection, lead to highly convergent synthetic designs, and more significantly, take advantage of the commercially available free glycans isolated from nature, which could considerably facilitate the synthesis of complex glycopeptides and glycoproteins. © 2005 Wiley Periodicals, Inc. [source] Induction of carbohydrate-specific antibodies in HLA-DR transgenic mice by a synthetic glycopeptide: a potential anti cancer vaccine for human useCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2003S. Vichier-Guerre Abstract: Over the last few years, anticancer immunotherapy has emerged as a new exciting area for controlling tumors. In particular, vaccination using synthetic tumor-associated antigens (TAA), such as carbohydrate antigens hold promise for generating a specific antitumor response by targeting the immune system to cancer cells. However, development of synthetic vaccines for human use is hampered by the extreme polymorphism of human leukocyte-associated antigens (HLA). In order to stimulate a T-cell dependent anticarbohydrate response, and to bypass the HLA polymorphism of the human population, we designed and synthesized a glycopeptide vaccine containing a cluster of a carbohydrate TAA B-cell epitope (Tn antigen: , -GalNAc-Ser) covalently linked to peptides corresponding to the Pan DR ,universal' T-helper epitope (PADRE) and to a cytotoxic T lymphocyte (CTL) epitope from the carcinoembryonic antigen (CEA). The immunogenicity of the construct was evaluated in outbred mice as well as in HLA transgenic mice (HLA-DR1, and HLA-DR4). A strong T-cell dependent antibody response specific for the Tn antigen was elicited in both outbred and HLA transgenic mice. The antibodies induced by the glycopeptide construct efficiently recognized a human tumor cell line underlying the biological relevance of the response. The rational design and synthesis of the glycopeptide construct presented herein, together with its efficacy to induce antibodies specific for native tumor carbohydrate antigens, demonstrate the potential of a such synthetic molecule as an anticancer vaccine candidate for human use. [source] FRET-Based Direct and Continuous Monitoring of Human Fucosyltransferases Activity: An Efficient synthesis of Versatile GDP- L -Fucose Derivatives from Abundant d- Galactose,CHEMISTRY - A EUROPEAN JOURNAL, Issue 2 2008Takahiro Maeda Abstract We have developed a facile and versatile protocol for the continuous monitoring of human fucosyltransferases activity by using fluorescence energy resonance transfer (FRET), and have explored the feasibility of its use in an inhibitor screening assay. A convenient sugar nucleotide with a fluorogenic probe, 6-deoxy-6- N -(2-naphalene-2-yl-acetamide)-,- L -galactopyranos-1-yl-guanosine 5,-diphosphate disodium salt (1), was efficiently synthesized from naturally abundant D -galactopyranose via a key intermediate, 6-azide-1,2,3,4-tetra- O -benzoyl-6-deoxy-,- L -galactopyranose (10). It was demonstrated that the combined use of the glycosyl donor 1 and a dansylated acceptor substrate, sialyl-,2,3-LacNAc derivative (2) allowed us to carry out highly sensitive, direct, and continuous in vitro monitoring of the generation of sialyl Lewis,X (SLex), which is catalyzed by human ,-1,3-fucosyltransferase,VI (FUT-VI). A kinetic analysis revealed that compound 1 was an excellent donor substrate (KM=0.94,,M and Vmax=0.14,,M,min,1) for detecting human FUT-VI activity. To the best of our knowledge, this synthetic fluorogenic probe is the most sensitive and selective donor substrate for FUT-VI among all of the known GDP-Fuc analogues, including the parent GDP-Fuc. When a dansylated asparagine-linked glycopeptide 20, which is derived from egg yolk was employed as an alternate acceptor substrate, a FRET-based assay with compound 1 could be used to directly monitor the ,1,6-fucosylation at the reducing terminal GlcNAc residue by human FUT-VIII (KM=175,,M and Vmax=0.06,,M/,min); this indicates that the present method might become a general protocol for the characterization of various mammalian fucosyltransferases in the presence of designated fluorogenic acceptor substrates. The present protocol revealed that compound 23, which was obtained by a 1,3-dipolar cycloaddition between the disodium salt 16 and 1-ethynyl-naphthalene exhibits highly potent inhibitory effects against the FUT-VI-mediated sialyl Lewis,X synthesis (IC50=5.4,,M). [source] Induction of a Melanoma-Specific Antibody Response by a Monovalent, but not a Divalent, Synthetic GM2 NeoglycopeptideCHEMMEDCHEM, Issue 4 2009S. Bay Dr. Abstract Human tumor cell-specific antibodies were induced in mice after immunization with a synthetic glycopeptide, which is based on the GM2 ganglioside carbohydrate moiety produced on a gram scale in bacteria. Such neoglycopeptides represent a promising cancer vaccine strategy for active immunotherapy targeting carbohydrates. The GM2 ganglioside represents an important target for specific anticancer immunotherapy. We designed and synthesized a neoglycopeptide immunogen displaying one or two copies of the GM2 tetrasaccharidic moiety. These glycopeptides were prepared using the Huisgen cycloaddition, which enables the efficient ligation of the alkyne-functionalized biosynthesized GM2 with an azido CD4+ T,cell epitope peptide. It is worth noting that the GM2 can be produced on a gram scale in bacteria, which can be advantageous for a scale-up of the process. We show here for the first time that a fully synthetic glycopeptide, which is based on a ganglioside carbohydrate moiety, can induce human tumor cell-specific antibodies after immunization in mice. Interestingly, the monovalent, but not the divalent, form of GM2 peptide construct induced antimelanoma antibodies. Unlike traditional vaccines, this vaccine is a pure chemically-defined entity, a key quality for consistent studies and safe clinical evaluation. Therefore, such carbohydrate,peptide conjugate represents a promising cancer vaccine strategy for active immunotherapy targeting gangliosides. [source] Additive concentration effects on enantioselective separations in supercritical fluid chromatography,CHIRALITY, Issue 4 2003Karen W. Phinney Abstract Polar additive concentration effects in supercritical fluid chromatography were studied on chiral stationary phases having either a macrocyclic glycopeptide or a derivatized polysaccharide as the chiral selector. Two basic additives, isopropylamine and triethylamine, were incorporated into the methanol modifier at various concentrations and the effects on retention, selectivity, and resolution were monitored. Many of the analytes failed to elute from the macrocyclic glycopeptide stationary phase in the absence of an additive and the most noticeable effect of increasing additive concentration was a significant decrease in retention. On the derivatized polysaccharide stationary phase the additives had little effect on retention, but they did foster significant improvements in peak shape and resolution. Chirality 15:287,294, 2003. Published 2003 Wiley-Liss, Inc. [source] The use and therapeutic drug monitoring of teicoplanin in the UKCLINICAL MICROBIOLOGY AND INFECTION, Issue 1 2004E. S. R. Darley Abstract Teicoplanin dosage recommendations for specific infections have been modified in recent years. However, there was no significant increase in the proportion of pre-dose concentrations >,20 mg/L between 1994 and 1998 in samples sent for teicoplanin assay at the Regional Antimicrobial Reference Laboratory, Bristol, UK. A questionnaire on the use of teicoplanin and therapeutic drug monitoring (TDM) was sent to all UK National External Quality Assurance Scheme antibiotic assay users. Teicoplanin was widely used in the UK, although vancomycin was more popular as a choice of glycopeptide. Fewer than 25% recommended teicoplanin TDM during routine use, the main reasons being perceived lack of toxicity and lack of evidence for the use of teicoplanin TDM. Pre-dose concentrations <,20 mg/L were considered appropriate for treatment of bacteraemia caused by methicillin-resistant Staphylococcus aureus by 53% of those responding. Data sheet advice was relied upon more than TDM as an indication of therapeutic dosing. Microbiologists who mainly used vancomycin tended to perform more TDM and seek higher serum concentrations when using teicoplanin than those who preferentially used teicoplanin. [source] Analysis of chicken and turkey ovalbumins by microchip electrophoresis combined with exoglycosidase digestionELECTROPHORESIS, Issue 18 2003Xiuli Mao Abstract The polypeptide and carbohydrate patterns of two glycoproteins, chicken ovalbumin (CO) and turkey ovalbumin (TO), were analyzed by microchip electrophoresis (ME), following digestion with proteases and exoglycosidases. Glycopeptides derived from ovalbumin were obtained by digestion with Pronase, followed by dialysis, and then separated by ME. Using CO as model, the method was developed to deduce the structure of glycans from glycoproteins by comparing the electropherograms of glycopeptides with and without digestion of exolycosidases. Applying the same approach, the structure of oligosaccharides linked to TO was determined. TO was found to contain high-mannose type oligosaccharides and oligosaccharides with terminal N -acetylglucosamine residues. The complete primary analysis of CO and TO by ME described in this paper provides a basis for an analysis of glycoproteins with an integrated microfluidic chip. [source] Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresisELECTROPHORESIS, Issue 16 2003Gianfranco Mamone Abstract We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five ,-, fifteen ,s1 -, ten ,s2 -, and four ,-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to ,-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single ,-CN component. The phosphate group on site Ser12 of tryptic peptide 8,22 of most phosphorylated ,s1 -CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two ,-CN components was determined by means of MS/MS analysis. [source] IPSE/alpha-1, a major secretory glycoprotein antigen from schistosome eggs, expresses the Lewis X motif on core-difucosylated N-glycansFEBS JOURNAL, Issue 10 2006Manfred Wuhrer Schistosomes are parasitic flatworms that infect millions of people in (sub)tropical areas around the world. Glycoconjugates of schistosomes play a critical role in the interaction of the different developmental stages of the parasite with the host. In particular, glycosylated components of the eggs produced by the adult worm pairs living in the bloodstream are strongly immunogenic. We have investigated the glycosylation of interleukin-4-inducing factor from schistosome eggs (IPSE/alpha-1), a major secretory egg antigen from Schistosoma mansoni that triggers interleukin-4 production in human basophils, by MS analysis of tryptic glycopeptides. Nanoscale LC-MS(/MS) and MALDI-TOF(/TOF)-MS studies combined with enzymatic degradations showed that monomeric IPSE/alpha-1 contains two N-glycosylation sites, which are each occupied for a large proportion with core-difucosylated diantennary glycans that carry one or more Lewis X motifs. Lewis X has been reported as a major immunogenic glycan element of schistosomes. This is the first report both on the expression of Lewis X on a specific schistosome egg protein and on a protein-specific glycosylation analysis of schistosome eggs. [source] Evolution of peptidoglycan biosynthesis under the selective pressure of antibiotics in Gram-positive bacteriaFEMS MICROBIOLOGY REVIEWS, Issue 2 2008Jean-Luc Mainardi Abstract Acquisition of resistance to the two classes of antibiotics therapeutically used against Gram-positive bacteria, the glycopeptides and the ,-lactams, has revealed an unexpected flexibility in the peptidoglycan assembly pathway. Glycopeptides select for diversification of the fifth position of stem pentapeptides because replacement of d -Ala by d -lactate or d -Ser at this position prevents binding of the drugs to peptidoglycan precursors. The substitution is generally well tolerated by the classical d,d -transpeptidases belonging to the penicillin-binding protein family, except by low-affinity enzymes. Total elimination of the fifth residue by a d,d -carboxypeptidase requires a novel cross-linking enzyme able to process the resulting tetrapeptide stems. This enzyme, an l,d -transpeptidase, confers cross-resistance to ,-lactams and glycopeptides. Diversification of the side chain of the precursors, presumably in response to the selective pressure of peptidoglycan endopeptidases, is controlled by aminoacyl transferases of the Fem family that redirect specific aminoacyl-tRNAs from translation to peptidoglycan synthesis. Diversification of the side chains has been accompanied by a parallel divergent evolution of the substrate specificity of the l,d -transpeptidases, in contrast to the d,d -transpeptidases, which display an unexpected broad specificity. This review focuses on the role of antibiotics in selecting or counter-selecting diversification of the structure of peptidoglycan precursors and their mode of polymerization. [source] Molecular pathology of NEU1 gene in sialidosis,HUMAN MUTATION, Issue 5 2003Volkan Seyrantepe Abstract Lysosomal sialidase (EC 3.2.1.18) has a dual physiological function; it participates in intralysosomal catabolism of sialylated glycoconjugates and is involved in cellular immune response. Mutations in the sialidase gene NEU1, located on chromosome 6p21.3, result in autosomal recessive disorder, sialidosis, which is characterized by the progressive lysosomal storage of sialylated glycopeptides and oligosaccharides. Sialidosis type I is a milder, late-onset, normosomatic form of the disorder. Type I patients develop visual defects, myoclonus syndrome, cherry-red macular spots, ataxia, hyperreflexia, and seizures. The severe early-onset form, sialidosis type II, is also associated with dysostosis multiplex, Hurler-like phenotype, mental retardation, and hepatosplenomegaly. We summarize information on the 34 unique mutations determined so far in the sialidase gene, including four novel missense and one novel nonsense mutations found in two Czech and two French sialidosis patients. The analysis of sialidase mutations in sialidosis revealed considerable molecular heterogeneity, reflecting the diversity of clinical phenotypes that make molecular diagnosis difficult. The majority of sialidosis patients have had missense mutations, many of which have been expressed; their effects on activity, stability, intracellular localization, and supramolecular organization of sialidase were studied. A structural model of sialidase allowed us to localize mutations in the sialidase molecule and to predict their impact on the tertiary structure and biochemical properties of the enzyme. Hum Mutat 22:343,352, 2003. © 2003 Wiley-Liss, Inc. [source] Glycan side chains on naturally presented MHC class II ligandsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2005Jörn Dengjel Abstract The molecular characterization of unknown naturally presented major histocompatibility complex (MHC) class II glycopeptides carrying complex glycans has so far not been achieved, reflecting the different fragmentation characteristics of sugars and peptides in mass spectrometric analysis. Human leukocyte antigen (HLA)-DR-bound peptides were isolated by affinity purification, separated via high performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. We were able to identify two naturally processed MHC class II ligands, CD53122,136 and CD53121,136, carrying complex N -linked glycan side chains by a combination of in-source and collision-induced fragmentation on a quadrupole time-of-flight tandem mass spectrometer. Copyright © 2005 John Wiley & Sons, Ltd. [source] Design and synthesis of new trehalose-conjugated pentapeptides as inhibitors of A,(1,42) fibrillogenesis and toxicity,JOURNAL OF PEPTIDE SCIENCE, Issue 3 2009Paolo De Bona Abstract Aggregation of the amyloid A, peptide and its accumulation into insoluble deposits (plaques) are believed to be the main cause of neuronal dysfunction associated with Alzheimer's disease (AD); small molecules that can interfere with the A, amyloid fibril formation are therefore of interest for a potential therapeutic strategy. Three new trehalose-conjugated peptides of the well known ,-sheet breaker peptide iA,5p, were synthesized. The disaccharide was covalently attached to different sites of the LPFFD peptide chain, i.e. at the N-terminus, C-terminus or at the Asp side chain. CD spectroscopy in different solvents was used to assess changes in the peptide conformation of these compounds. The effects of these glycopeptides on the self-assembly and morphology of A, aggregates were investigated by ThT fluorescence assay and dynamic Scanning Force Microscopy, respectively. All the synthesized compounds were tested as inhibitors of A, toxicity toward pure cultures of rat cortical neurons. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] Characterization of sialylated and fucosylated glycopeptides of ,2-glycoprotein I by a combination of HILIC LC and MALDI MS/MSJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010Akira Kondo Abstract Characterization of low microgram levels of glycoprotein remains a challenge due to extensive heterogeneity of the conjugated N -glycans at each individual glycosylation site. We present an optimized, sensitive workflow for glycopeptide isolation and characterization that exploits the complementary features of RP (Poros R2) and hydrophilic (zwitter-ionic hydrophilic interaction chromatography) chromatographic resins. The glycopeptide analysis workflow was applied to human ,2-glycoprotein I (,2-GPI, apolipoprotein H), which contains multiple N -glycosylation sites. Conditions for rapid proteolytic digestion of ,2-GPI using low-specificity proteases were optimized to detect ,2-GPI glycopeptides by MS. We demonstrate the importance of ensuring sufficient column capacity of both hydrophobic and hydrophilic stationary phases for optimal glycoprofiling by MS. The enriched glycopeptides were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and tri-antennary complex type glycans, were characterized at three N -glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of ,2-GPI. Further exploration of the complementary nature of RP and HILIC stationary phases for glycopeptide isolation prior to MS analysis may eventually enable systematic analysis of complex glycoprotein samples in functional proteomic research and advance our understanding of the biological role of protein glycosylation. [source] Protein glycosylation analysis by HILIC-LC-MS of Proteinase K-generated N - and O -glycopeptidesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 6-7 2010Gerhild Zauner Abstract Analysis of protein glycosylation is essential in order to correlate certain disease types with oligosaccharide structures on proteins. Here, a method for the MS characterization of site-specific protein glycosylation is presented. Using asialofetuin and fetuin as model substances, a protocol for glycopeptide dissection was developed based on unspecific proteolysis by Proteinase K. The resulting glycopeptides were then resolved by nanoscale hydrophilic interaction liquid chromatography-electrospray multistage MS. The early elution range of O -glycopeptides was clearly separated from the late elution range of N -glycopeptides. Glycopeptides were analyzed by ion trap-MS/MS, which revealed fragmentations of glycosidic linkages and some peptide backbone cleavages; MS3 spectra predominantly exhibited cleavages of the peptide backbone and provided essential information on the peptide sequence. The previously reported N - and O -glycan attachment sites of fetuin could be confirmed; moreover using our method, the occupation of a new, additional O -glycosylation site serine 296 was found. In conclusion, this approach appears to be a valuable technique for in-depth analysis of the site-specific N -glycosylation and O -glycosylation of individual glycoproteins. [source] Manipulation of Electrostatic and Saccharide Linker Interactions in the Design of Efficient Glycopolypeptide-Based Cholera Toxin InhibitorsMACROMOLECULAR BIOSCIENCE, Issue 1 2010Ronak Maheshwari Abstract Multivalent, glycopolymer inhibitors designed for the treatment of disease and pathogen infection have shown improvements in binding correlated with general changes in glycopolymer architecture and composition. We have previously demonstrated that control of glycopolypeptide backbone extension and ligand spacing significantly impacts the inhibition of the cholera toxin B subunit pentamer (CT B5) by these polymers. In the studies reported here, we elucidate the role of backbone charge and linker length in modulating the inhibition event. Peptides of the sequence AXPXG (where X is a positive, neutral or negative amino acid), equipped with the alkyne functionality of propargyl glycine, were designed and synthesized via solid-phase peptide synthetic methods and glycosylated via Cu(I)-catalyzed alkyne-azide cycloaddition reactions. The capacity of the glycopeptides to inhibit the binding of the B5 subunit of cholera toxin was evaluated. These studies indicated that glycopeptides with a negatively charged backbone show improved inhibition of the binding event relative to the other glycopeptides. In addition, variations in the length of the linker between the peptide and the saccharide ligand also affected the inhibition of CT by the glycopeptides. Our findings suggest that, apart from appropriate saccharide spacing and polypeptide chain extension, saccharide linker conformation and the systematic placement of charges on the polypeptide backbone are also significant variables that can be tuned to improve the inhibitory potencies of glycopolypeptide-based multivalent inhibitors. [source] Glycopeptide and glycoprotein synthesis involving unprotected carbohydrate building blocks,MEDICINAL RESEARCH REVIEWS, Issue 6 2005Zhongwu Guo Abstract This review summarizes the chemical and chemoenzymatic synthesis of glycopeptides and glycoproteins using unprotected carbohydrates as key intermediates. The synthetic methods covered herein include the convergent synthesis of glycopeptides by chemoselective ligation of peptides and free glycans, solution- and solid-phase synthesis of glycopeptides by sequential peptide elongation with unprotected glycosyl amino acids or short glycopeptides as building blocks, and the synthesis of glycopeptides by enzymatic and/or chemical elongation of the free glycans. The use of unprotected carbohydrates in these syntheses can circumvent the final-stage carbohydrate deprotection, lead to highly convergent synthetic designs, and more significantly, take advantage of the commercially available free glycans isolated from nature, which could considerably facilitate the synthesis of complex glycopeptides and glycoproteins. © 2005 Wiley Periodicals, Inc. [source] A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faeciumMOLECULAR MICROBIOLOGY, Issue 3 2003Florence Depardieu Summary Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB / vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d -alanine: d -alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSB, . The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanS B, histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanS B , VanS B, and VanR B proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanS B and VanS B, histidine kinases and phosphotransfer to the VanR B response regulator did not differ significantly. However, VanS B, was deficient in VanR B phosphatase activity leading to accumulation of phosphorylated VanR B . Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d -alanyl- d -alanine ending pentapeptide and to constitutive synthesis of d -alanyl- d -lactate terminating peptidoglycan precursors, following loss of d -alanine: d -alanine ligase and of VanS B phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a ,slippage' type of genetic rearrangement in VanB-type strains. [source] Trends in antimicrobial utilization at a Spanish general hospital during a 5-year periodPHARMACOEPIDEMIOLOGY AND DRUG SAFETY, Issue 3 2003Lorena Hermosilla Nájera Abstract Purpose Antimicrobials are a major part of hospital pharmacy budgets and must be considered in resource planning and spending projections. This study describes the profile of antibiotic use at a medium-sized hospital (by examining the ICU separately) and analyses its evolution over the period 1996,2000. Methods Descriptive and retrospective study. Pharmacy records were reviewed to identify oral and parenteral antimicrobial agents administered to inpatients. Results were expressed in Daily Defined Doses (DDD) per 100 stays and day. Results During the 5-year study period 176.162 DDD/100 s-d of antibiotics were consumed in the ICU, whereas in the rest of the hospital usage was much lower (54.438 DDD/100 s-d). Aminoglycosides, cephalosporins, penicillins, glycopeptides and carbapenems were the most commonly used groups of antimicrobials in the ICU, and penicillins, cephalosporins, trimethoprim/sulfonamide combinations, aminoglycosides and quinolones in the rest of the hospital. Conclusions ICUs have some special features which make them different to the other inpatient areas. Because of that fact we consider it important to study this specific patient-care area separately. Copyright © 2003 John Wiley & Sons, Ltd. [source] On-plate-selective enrichment of glycopeptides using boronic acid-modified gold nanoparticles for direct MALDI-QIT-TOF MS analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2009Jia Tang Abstract In this study, an on-plate-selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a stainless-steel plate, then modified with 4-mercaptophenylboronic acid to provide porous substrate with large specific surface and dual functions. These spots were used to selectively capture glycopeptides from peptide mixtures and the captured target peptides could be analyzed by MALDI-MS simply by deposition of 2,5-dihydroxybenzoic acid matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency. In this way, the enrichment, washing and detection steps can all be fulfilled on a single MALDI target plate. The relatively small sample amount needed, low detection limit and rapid selective enrichment have made this on-plate strategy promising for online enrichment of glycopeptides, which could be applied in high-throughput proteome research. [source] Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantificationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2009Jennifer J. Hill Dr. Abstract Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label-free MS-based approach to identify formerly N -linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to ,2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine,protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments. [source] Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharidesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2008Johannes Stadlmann Abstract Two LC-ESI-MS methods for the analysis of antibody glycosylation are presented. In the first approach, tryptic glycopeptides are separated by RP chromatography and analyzed by ESI-MS. This "glycopeptide strategy" allows a protein- and subclass-specific quantitation of both neutral and sialylated glycan structures. Additional information about under- or deglycosylation and the protein backbone, e.g., termini, can be extracted from the same data. In the second LC-ESI-MS method, released oligosaccharides are separated on porous graphitic carbon (PGC). A complete structural assignment of neutral and sialylated oligosaccharides occurring on antibodies is thereby achieved in one chromatographic run. The two methods were applied to polyclonal human IgG, to commercial mAb expressed in CHO cells (Rituximab, Xolair, and Herceptin), in SP2/0 (Erbitux and Remicade) or NS0 cells (Zenapax) and the anti-HIV antibody 4E10 produced either in CHO cells or in a human cell line. Both methods require comparably little sample preparation and can be applied to SDS-PAGE bands. They both outperform non-MS methods in terms of reliability of peak assignment and MALDI-MS of underivatized glycans with regard to the recording of sialylated structures. Regarding fast and yet detailed structural assignment, LC-MS on graphitic carbon supersedes all other current methods. [source] Determination of the site-specific and isoform-specific glycosylation in human plasma-derived antithrombin by IEF and capillary HPLC-ESI-MS/MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2005Alexander Plematl Abstract The glycan structures of the major and more than ten minor populated isoforms of antithrombin (AT) were determined after separation of the isoforms by IEF using IPG strips. The bands excised from the gel were reduced, derivatized by iodoacetamide and submitted to tryptic digestion. The digest was analyzed by RP-HPLC-ESI-MS equipped with a quadrupole ion-trap mass analyzer. MS/MS experiments allowed establishing the monosaccharide compositions in the glycopeptides. For the major isoform of ,-AT four identical biantennary glycans with two terminal sialic acids (SA) each, a total of eight SA, were found in full agreement with the literature. In the IEF-band containing this major isoform (pI 5.18) a further, much less abundant, isoform was detected showing a fucosylation on the glycan attached to Asn155 but being of otherwise identical structure as described above. The isoforms with pI 5.10 were found to include one triantennary glycan, all antennas carrying terminal SA. The occurrence of triantennary structure is site specific, involving the peptides with Asn135 and Asn155, alternately. At pI 5.24 we found those four isoforms that carry the glycans like the main-isoform of ,-AT but missing one terminal SA. There was no site specificity found for the mono-sialo structure. The isoform at pI 5.31 is the major isoform of ,-AT containing three identical biantennary structures being fully sialylated. No isoforms (above 0.5% abundance) with two glycans only or three glycans other than ,-AT were detected. Fucosylation was found in the main isoform with an abundance of about 5%, and as expected with all the other isoforms with a comparable abundance. [source] A mass spectrometric strategy for profiling glycoproteinoses, Pompe disease, and sialic acid storage diseasesPROTEOMICS - CLINICAL APPLICATIONS, Issue 4 2008Valegh Faid Abstract Glycoproteinoses, Pompe disease, and sialic acid storage diseases are characterized by a massive accumulation of unprocessed oligosaccharides and/or glycoconjugates in urine. The identification of these glycocompounds is essential for a proper diagnosis. In this study, we investigated the potential of MALDI-TOF-MS to identify glycocompounds present in urine from patients with different inborn errors of glycan metabolism. Urinary glycocompounds were permethylated, and analyzed using GC-MS and MALDI-TOF-MS. In order to confirm tentative assignments, a second aliquot of urine was purified on a C18 Sep-Pak cartridge and glycocompounds were desalted on a column of nonporous graphitized carbon. The glycocompounds were then sequentially on-plate digested using an array of exoglycosidases. A range of disease-specific oligosaccharides as well as glycopeptides was identified for all oligosacchariduria models. In addition, free sialic acid accumulated in urine from a patient suffering from French-type sialuria, has been detected by a GC-MS approach, which could be applied to other sialic acid storage diseases. This procedure is simple, and can be performed in few simple steps in less than 24,h. This current method can be applied for newborn screening for other inherited metabolic diseases as well as for assessing treatments in clinical trials. [source] Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2009William R. Alley Jr. Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip-based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C18 LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C18 chips. On the other hand, hydrophobic glycopeptides were better retained on C18 chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1,pmol). The performance of both media also appeared comparable when analyzing a four-protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase. Copyright © 2009 John Wiley & Sons, Ltd. [source] Complementary structural information of positive- and negative-ion MSn spectra of glycopeptides with neutral and sialylated N-glycansRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2006Kisaburo Deguchi Positive- and negative-ion MSn spectra of chicken egg yolk glycopeptides binding a neutral and a sialylated N-glycan were acquired by using electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) and collision-induced dissociation (CID) with helium as collision gas. Several characteristic differences were observed between the positive- and negative-ion CID MSn (n,=,2, 3) spectra. In the positive-ion MS2 spectra, the peptide moiety was presumably stable, but the neutral N-glycan moiety caused several B-type fragmentations and the sialylated N-glycan almost lost sialic acid(s). In contrast, in the negative-ion MS2 spectra, the peptide moiety caused several side-chain and N-glycan residue (e.g., N -acetylglucosamine (GlcNAc) residue) fragmentations in addition to backbone cleavages, but the N-glycan moieties were relatively stable. The positive-ion MS3 spectra derived from the protonated peptide ion containing a GlcNAc residue (203.1,Da) provided enough information to determine the peptide amino-acid sequence including the glycosylation site, while the negative-ion MS3 spectra derived from the deprotonated peptide containing a 0,2X1 -type cross-ring cleavage (83.1,Da) complicated the peptide sequence analysis due to side-chain and 0,2X1 residue related fragmentations. However, for the structural information of the N-glycan moiety of the glycopeptides, the negative-ion CID MS3 spectra derived from the deprotonated 2,4A6 -type cross-ring cleavage ion (neutral N-glycan) or the doubly deprotonated B6 -type fragment ion (sialylated N-glycan) are more informative than are those of the corresponding positive-ion CID MS3 spectra. Thus, the positive-ion mode of CID is useful for the analyses of peptide amino-acid sequences including the glycosylation site. The negative-ion mode of CID is especially useful for sialylated N-glycan structural analysis. Therefore, in the structural analysis of N-glycopeptides, their roles are complementary. Copyright © 2006 John Wiley & Sons, Ltd. [source] Identification of substituted sites on MUC5AC mucin motif peptides after enzymatic O-glycosylation combining ,-elimination and fixed-charge derivatizationRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 1 2002X. Czeszak A strategy for determination of O-glycosylation site(s) in glycopeptides has been developed using model compounds obtained by enzymatic glycosylation (by human GaNTase-T2 isoform) on peptides derived from the human MUC5AC mucin tandem repeat motif. The ,-elimination-addition reaction (using dimethylamine and concomitantly ethanethiol) on the formerly glycosylated sites through a Michael-type condensation produced efficient deglycosylation with appropriate chemical modification. After N-terminal derivatization by a phosphonium group, peptide sequencing was then carried out by nanospray tandem mass spectrometry experiments. The highly predictable fragmentation pathways of these fixed-charge phosphonium derivatives enable straightforward recognition of glycosylation site(s) based on the mass increment of +44,Da for originally glycosylated threonine compared to the mass of fragments containing nonglycosylated residues. Copyright ©,2001 John Wiley & Sons, Ltd. [source] Rapid and sensitive quantitation of antibiotics in fermentations by electrospray mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2001Janine Morgan Electrospray ionisation mass spectrometry (ESI-MS) has been used for the determination and quantitation of a broad range of 24 antibiotics, from groups including aminoglycosides, ,-lactams, tetracyclines, antifungals and glycopeptides. Spectra have been acquired for all 24 antibiotics derived from pure samples dissolved in acetonitrile/water, along with samples extracted from complex fermentation liquor. Quantitation was carried out by the detection of the protonated molecules, using time-scheduled single-ion monitoring (SIM). ESI-MS was used to detect and quantify to 5-µM levels. A one-step extraction of antibiotics with an organic solvent (methanol) was used for this rapid and simple procedure. Specificity is not matched by other methods and antibiotic analogues (e.g. the five forms of erythromycin) can be determined within minutes. Copyright © 2001 John Wiley & Sons, Ltd. [source] Effects of glycopeptides on development, growth and non-specific immunity of pearl oyster Pinctada fucata (Gould)AQUACULTURE NUTRITION, Issue 5 2010S. ZHANG Abstract The effects of glycopeptides, prepared from pearl oyster Pinctada fucata, on embryonic development, larval and juvenile growth and adult non-specific immunity of P. fucata were investigated in this study. Glycopeptides had a pronounced stimulatory effect on embryonic development and larval and juvenile growth of P. fucata. enhancing with increased glycopeptide concentrations. All of haemocytes, phagocytosis, aggregation, serum microbiostatic activity and bacteriocidal activity all showed significant increases after 60-day feeding, relative to unfed controls. The major conclusion is that glycopeptides had a pronounced stimulatory effect on the non-specific immunity of pearl oysters. [source] Analysis of glycopeptide antibiotics using micellar electrokinetic chromatography and borate complexationBIOMEDICAL CHROMATOGRAPHY, Issue 2-3 2003Carmelle Lucas Abstract Micellar electrokinetic chromatography (MEKC) was investigated as a technique for the separation and analysis of the following related glycopeptide antibiotics: ,-avoparcin, ,-avoparcin, ristocetin A, ristocetin B and vancomycin. Sodium dodecyl sulfate (SDS) micelles were employed as the pseudostationary phase in conjunction with borate or CHES buffers at pH 9.2. A complete separation of the glycopeptides was achieved only when two separation mechanisms were employed simultaneously: (i) differential partitioning of the glycopeptides into SDS micelles; and (ii) differential complexation of the glycopeptides with the borate anion from the borate buffer. Quantitatively, linearity was confirmed for each antibiotic from 0.5 to 40,ppm, with correlation coefficients (r2) ranging from 0.9996 (vancomycin and ,-avoparcin) to 0.9986 (,-avoparcin). Detection limits ranging from 0.01,ppm (vancomycin) to 0.2,ppm (avoparcin) were achieved, and the mean recovery of avoparcin at the 10,ppm level was 99.2%. Copyright © 2003 John Wiley & Sons, Ltd. [source] |