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Nancy Leymarie,
Paula J Griffin,
Karen Jonscher,
Daniel Kolarich,
Ron Orlando,
Mark McComb,
Joseph Zaia,
Jennifer Aguilan,
William R Alley,
Friederich Altmann, [......],
Rosa I Viner,
Yanming An,
Leena Valmu,
Yoshinao Wada,
Megan Watson,
Markus Windwarder,
Randy Whittal,
Manfred Wuhrer,
Yiying Zhu,
Chunxia Zou
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ABSTRACT: One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. Accurate comparison of glycoforms and quantification of glycosites is an essential step in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group (gPRG) study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked and it has been observed that prostate cancer tissues and cell lines, contain more antennae than the benign form. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact use of PSA as a biomarker. We studied standard peptide based proteomics/glycomics methodologies including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences in glycoforms between two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods, respectively.
Molecular & Cellular Proteomics 06/2013; · 7.40 Impact Factor
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ABSTRACT: All Campylobacter species are capable of N-glycosylating their proteins and releasing the same oligosaccharides into the periplasm as free oligosaccharides (fOS). Previously, analysis of fOS production in Campylobacter required fOS derivatization or large culture volumes and several chromatography steps prior to fOS analysis. In this study, label-free fOS extraction and purification methods were developed and coupled with quantitative analysis techniques. Our method follows three simple steps: I) fOS extraction from the periplasmic space, II) fOS purification using silica gel chromatography followed by porous graphitized carbon purification and III) fOS analysis and accurate quantitation using a combination of thin-layer chromatography, mass spectrometry, NMR and high performance anion exchange chromatography with pulsed amperometric detection. We applied our techniques to analyse fOS from C. jejuni, C. lari, C. rectus and C. fetus fetus that produce different fOS structures. We accurately quantified fOS in Campylobacter species that ranged from 7.80 (± 0.84) to 49.82 (± 0.46) nmoles per gram of wet cell pellet and determined that the C. jejuni fOS comprises 2.5% of the dry cell weight. In addition, a novel di-phosphorylated fOS species was identified in C. lari. This method provides a sensitive and quantitative method to investigate the genesis, biology and breakdown of fOS in the bacterial N-glycosylation systems.
Biopolymers 06/2013; · 2.87 Impact Factor
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Jeremy A Iwashkiw,
Andrea Seper,
Brent S Weber,
Nichollas E Scott,
Evgeny Vinogradov,
Chad Stratilo, Bela Reiz,
Stuart J Cordwell,
Randy Whittal,
Stefan Schild,
Mario F Feldman
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ABSTRACT: Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening "superbugs" for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.
PLoS Pathogens 06/2012; 8(6):e1002758. · 9.13 Impact Factor
