The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Henning Urlaub - One of the best experts on this subject based on the ideXlab platform.
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evaluation and optimization of high field asymmetric waveform ion mobility spectrometry for multiplexed quantitative site specific n glycoproteomics
Analytical Chemistry, 2021Co-Authors: Pan Fang, Ivan Silbern, Thomas Oellerich, Kuan-ting Pan, Henning Urlaub, Rosa VinerAbstract:The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has been shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size, and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 methods provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass spectrometry-based qualitative and quantitative N-glycoproteomics.
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Evaluation and Optimization of High-Field Asymmetric Waveform Ion Mobility Spectrometry for Multiplexed Quantitative Site-specific N-glycoproteomics
2021Co-Authors: Pan Fang, Ivan Silbern, Viner R, Thomas Oellerich, Kuan-ting Pan, Henning UrlaubAbstract:The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified IgM protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N-glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size and precursor m/z. Importantly, FAIMS also improved multiplexed N-glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass-spectrometry-based qualitative and quantitative N-glycoproteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/436434v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@126b92forg.highwire.dtl.DTLVardef@147d6e5org.highwire.dtl.DTLVardef@16d6eb0org.highwire.dtl.DTLVardef@17dfe2a_HPS_FORMAT_FIGEXP M_FIG C_FIG
Hansjoachim Gabius - One of the best experts on this subject based on the ideXlab platform.
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Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin‑3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides
2015Co-Authors: Maria C. Rodriguez, Hansjoachim Gabius, Svetlana Yegorova, Jean-philippe Pitteloud, Anais E. Chavaroche, Sabine André, Ana Ardá, Dimitriy Minond, Jesús Jiménez-barbero, Mare CudicAbstract:A shift to short-chain glycans is an observed change in mucin-type O-glycosylation in premalignant and malignant epithelia. Given the evidence that human galectin-3 can interact with mucins and also weakly with free tumor-associated Thomsen-Friedenreich (TF) antigen (CD176), the study of its interaction with MUC1 (glyco)peptides is of biomedical relevance. Glycosylated MUC1 fragments that carry the TF antigen attached through either Thr or Ser side chains were synthesized using standard Fmoc-based automated solid-phase peptide chemistry. The dissociation constants (Kd) for interaction of galectin-3 and the glycosylated MUC1 fragments measured by isothermal titration calorimetry decreased up to 10 times in comparison to that of the free TF disaccharide. No binding was observed for the nonglycosylated control version of the MUC1 peptide. The most notable feature of the binding of MUC1 glycopeptides to galectin-3 was a shift from a favorable enthalpy to an entropy-driven binding process. The comparatively diminished enthalpy contribution to the free energy (ΔG) was compensated by a considerable gain in the entropic term. 1H–15N heteronuclear single-quantum coherence spectroscopy nuclear magnetic resonance data reveal contact at the canonical site mainly by the glycan moiety of the MUC1 glycopeptide. Ligand-dependent differences in binding affinities were also confirmed by a novel assay for screening of low-affinity glycan–lectin interactions based on AlphaScreen technology. Another key finding is that the glycosylated MUC1 peptides exhibited activity in a concentration-dependent manner in cell-based assays revealing selectivity among human galectins. Thus, the presentation of this tumor-associated carbohydrate ligand by the natural peptide scaffold enhances its affinity, highlighting the significance of model studies of human lectins with synthetic glycopeptides
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single site mutational engineering and following monopegylation of the human lectin galectin 2 effects on ligand binding functional aspects and clearance from serum
Molecular Pharmaceutics, 2013Co-Authors: Jurgen Kopitz, Zdenek Fik, Sabine Andre, Karel Smetana, Hansjoachim GabiusAbstract:The emerging insights into the physiological significance of endogenous lectins prompted us to characterize the effect of monosubstitution with poly(ethylene glycol) (PEG; 5 kDa) on a human lectin. As role model, we used a member of the galectin family, that is, galectin-2, the Cys57Met (single-site) mutant and its monoPEGylated derivative. The activities of these three proteins were comparatively studied by biochemical, cell biological, and histochemical methods, using surface-immobilized glycoproteins, different types of cells presenting gangliosides or (glyco)proteins as counterreceptors in vitro and tissue sections. PEGylation led to decreases in affinity/signal intensity with context dependence. The introduction of the mutation, too, can influence reactivity. Assays on haemagglutination and inhibition of cell proliferation underscored that mutational engineering and substitution can (but must not necessarily) affect this protein's activity. Serum clearance in rats was markedly retarded by PEGylation. Overall, the bulky substitution, spatially comparable to N-glycans, can markedly reduce binding of the galectin to physiological binding sites.
Mulin Chen - One of the best experts on this subject based on the ideXlab platform.
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fabrication of highly stable glyco gold nanoparticles and development of a glyco gold nanoparticle based oriented immobilized antibody microarray for lectin goal assay
Chemistry: A European Journal, 2015Co-Authors: Lide Huang, Arup Kumar Adak, Chingching Yu, Weichen Hsiao, Mulin ChenAbstract:: The design of high-affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco-gold nanoparticles (glyco-AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco-AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70 mol %) and an amphiphilic linker (30 mol %) provide long-term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco-AuNPs enable the detection of model plant lectins such as Concanavalin A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop in situ glyco-AuNPs-based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that Glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of Glyconanoparticle-based biosensors.
Andre M Deelder - One of the best experts on this subject based on the ideXlab platform.
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n glycosylation profiling of colorectal cancer cell lines reveals association of fucosylation with differentiation and caudal type homebox 1 cdx1 villin mrna expression
Molecular & Cellular Proteomics, 2016Co-Authors: Stephanie Holst, Andre M Deelder, Carolien A M Koeleman, Anna J M Deuss, Gabi W Van Pelt, Sandra J Van Vliet, Juan J Garciavallejo, Wilma E Mesker, Rob A E M Tollenaar, Yoann RomboutsAbstract:Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.
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glycoproteomics based on tandem mass spectrometry of glycopeptides
Journal of Chromatography B, 2007Co-Authors: Manfred Wuhrer, Isabel M Catalina, Andre M Deelder, Cornelis H HokkeAbstract:Next to the identification of proteins and the determination of their expression levels, the analysis of post-translational modifications (PTM) is becoming an increasingly important aspect in proteomics. Here, we review mass spectrometric (MS) techniques for the study of protein glycosylation at the glycopeptide level. Enrichment and separation techniques for glycoproteins and glycopeptides from complex (glyco-)protein mixtures and digests are summarized. Various tandem MS (MS/MS) techniques for the analysis of glycopeptides are described and compared with respect to the information they provide on peptide sequence, glycan attachment site and glycan structure. Approaches using electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) of glycopeptides are presented and the following fragmentation techniques in glycopeptide analysis are compared: collision-induced fragmentation on different types of instruments, metastable fragmentation after MALDI ionization, infrared multi-photon dissociation, electron-capture dissociation and electron-transfer dissociation. This review discusses the potential and limitations of tandem mass spectrometry of glycopeptides as a tool in structural glycoproteomics.
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protein glycosylation analyzed by normal phase nano liquid chromatography mass spectrometry of glycopeptides
Analytical Chemistry, 2005Co-Authors: Manfred Wuhrer, Carolien A M Koeleman, And Cornelis H Hokke, Andre M DeelderAbstract:A new method for the mass spectrometric characterization of site-specific protein glycosylation is presented. Glycoprotein samples were subjected to unspecific proteolysis by Pronase, resulting in glycopeptides with peptide moieties of mostly two to eight amino acids. Resulting (glyco-)peptide samples were resolved by nanoscale normal-phase liquid chromatography (LC)−online mass spectrometry (MS). Retention depended on the size of the glycan chain and allowed the separation of identical peptide moieties containing different N-glycan structures. Glycopeptides were analyzed in an ion trap instrument performing repetitive ion isolation/fragmentation cycles. While the MS/MS spectra were dominated by fragmentations of glycosidic linkages, MS3 spectra exhibited cleavages of the peptide backbone and provided information on the peptide sequence and glycan attachment site. When applied to the model glycoproteins ribonuclease B and horseradish peroxidase (HRP), the method provided detailed insights into protein gly...
Jerry Eichler - One of the best experts on this subject based on the ideXlab platform.
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glyco engineering in archaea differential n glycosylation of the s layer glycoprotein in a transformed haloferax volcanii strain
Microbial Biotechnology, 2011Co-Authors: Doron Calo, Ziqiang Guan, Jerry EichlerAbstract:Summary Archaeal glycoproteins present a variety of N-linked glycans not seen elsewhere. The ability to harness the agents responsible for this unparalleled diversity offers the possibility of generating glycoproteins bearing tailored glycans, optimized for specific func- tions. With a well-defined N-glycosylation pathway and available genetic tools, the haloarchaeon Halof- erax volcanii represents a suitable platform for such glyco-engineering efforts. In Hfx. volcanii, the S- layer glycoprotein is modified by an N-linked pen- tasaccharide. In the following, S-layer glycoprotein N-glycosylationwasconsideredincellsinwhichAglD, the dolichol phosphate mannose synthase involved in addition of the final residue of the pentasaccharide, was replaced by a haloarchaeal homologue of AglJ, the enzyme involved in addition of the first residue of the N-linked pentasaccharide. In the engineering strain, the S-layer glycoprotein is modified by a novel N-linked glycan not found on this reporter from the parent strain. Moreover, deletion of AglD alone and introduction of the AglJ homologue from Halobacte- rium salinarum, OE2528R, into the deletion strain resulted in increased biosynthesis of the novel 894 Da glycan concomitant with reduced biogenesis of the pentasaccharide normally N-linked to the S-layer gly- coprotein. These findings justify efforts designed to transform Hfx. volcanii into a glyco-engineering 'workshop'.
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Glyco-engineering in Archaea: differential N-glycosylation of the Slayer glycoprotein in a transformed Haloferax volcanii strain. Microb Biotechnol 4: 461–470. doi: 10. 1111/j.1751-7915.2011.00250.x PMID: 21338478
2011Co-Authors: Doron Calo, Ziqiang Guan, Jerry Eichler, Department Of Life SciencesAbstract:glycans not seen elsewhere. The ability to harness the agents responsible for this unparalleled diversity offers the possibility of generating glycoproteins bearing tailored glycans, optimized for specific func-tions. With a well-defined N-glycosylation pathway and available genetic tools, the haloarchaeon Halof-erax volcanii represents a suitable platform for such glyco-engineering efforts. In Hfx. volcanii, the S-layer glycoprotein is modified by an N-linked pen-tasaccharide. In the following, S-layer glycoprotein N-glycosylation was considered in cells in which AglD, the dolichol phosphate mannose synthase involved in addition of the final residue of the pentasaccharide, was replaced by a haloarchaeal homologue of AglJ, the enzyme involved in addition of the first residue of the N-linked pentasaccharide. In the engineering strain, the S-layer glycoprotein is modified by a novel N-linked glycan not found on this reporter from the parent strain. Moreover, deletion of AglD alone and introduction of the AglJ homologue from Halobacte-rium salinarum, OE2528R, into the deletion strain resulted in increased biosynthesis of the novel 894 Da glycan concomitant with reduced biogenesis of the pentasaccharide normally N-linked to the S-layer gly-coprotein. These findings justify efforts designed to transform Hfx. volcanii into a glyco-engineering ‘workshop’