The Experts below are selected from a list of 322878 Experts worldwide ranked by ideXlab platform
Nico Callewaert - One of the best experts on this subject based on the ideXlab platform.
-
prostate protein n glycosylation profiling by means of DNA Sequencer assisted fluorophore assisted carbohydrate electrophoresis
Methods of Molecular Biology, 2019Co-Authors: Tijl Vermassen, Nico Callewaert, Sylvie Rottey, Joris DelangheAbstract:DNA Sequencer-assisted fluorophore-assisted carbohydrate electrophoresis allows for accurate profiling of the asparagine-linked (N-) glycosylation patterns, a posttranslational modification present on many soluble and membrane proteins. This technique has been extensively tested to identify N-glycosylation patterns associated with serum proteins. Here we describe the use of DNA Sequencer-assisted fluorophore-assisted carbohydrate electrophoresis to identify the N-glycosylation patterns of prostate proteins in urine.
-
Improved sample preparation for CE-LIF analysis of plant N-glycans.
Electrophoresis, 2011Co-Authors: Bieke Nagels, Francis Santens, Koen Weterings, Els J. M. Van Damme, Nico CallewaertAbstract:: In view of glycomics studies in plants, it is important to have sensitive tools that allow one to analyze and characterize the N-glycans present on plant proteins in different species. Earlier methods combined plant-based sample preparations with CE-LIF N-glycan analysis but suffered from background contaminations, often resulting in non-reproducible results. This publication describes a reproducible and sensitive protocol for the preparation and analysis of plant N-glycans, based on a combination of the 'in-gel release method' and N-glycan analysis on a multicapillary DNA Sequencer. Our protocol makes it possible to analyze plant N-glycans starting from low amounts of plant material with highly reproducible results. The developed protocol was validated for different plant species and plant cells.
-
glycofibrotest is a highly performant liver fibrosis biomarker derived from DNA Sequencer based serum protein glycomics
Molecular & Cellular Proteomics, 2009Co-Authors: Wouter Laroy, Joris Delanghe, Dieter Vanderschaeghe, Erwin Sablon, Philippe Halfon, Annelies Van Hecke, Nico CallewaertAbstract:Liver fibrosis is currently assessed by liver biopsy, a costly and rather cumbersome procedure that is unsuitable for frequent patient monitoring, which drives research into biomarkers for this purpose. To investigate whether the serum N-glycome contains information suitable for this goal, we developed a 96-well plate-based serum N-glycomics sample preparation protocol that only involves fluid transfer steps and incubations in a PCR thermocycler yielding 8-aminopyrene-1,3,6-trisulfonic acid-labeled N-glycans. These N-glycans are then ready for analysis on the capillary electrophoresis-based DNA Sequencers that are the current standard in clinical genetics laboratories worldwide. Subsequently we performed a multicenter, blinded study of 376 consecutive chronic hepatitis C virus patients for which liver biopsies and extensive serum biochemistry data were available. Among patients, the METAVIR fibrosis stage distribution was as follows: 10.6% F0, 44.4% F1, 20.5% F2, 18.4% F3, and 6.1% F4. We found that the ratio of two N-glycans, here called GlycoFibroTest, correlates with the histological fibrosis stage equally well as FibroTest (rho = 0.4-0.5 in F1-F4), which is used in the clinic today. Finally using affinity chromatography we depleted sera of immunoglobulin G, and this resulted in a complete removal of the undergalactosylated biantennary glycans from the N-glycome, which are partially determining GlycoFibroTest.
-
high throughput quantitative analysis of plant n glycan using a DNA Sequencer
Biochemical and Biophysical Research Communications, 2009Co-Authors: Nico Callewaert, Kyung Jin Lee, Jinhee Jung, Jung Mi Lee, Ohsuk Kwon, Hyun KangAbstract:Abstract High-throughput quantitative analytical method for plant N -glycan has been developed. All steps, including peptide N -glycosidase (PNGase) A treatment, glycan preparation, and exoglycosidase digestion, were optimized for high-throughput applications using 96-well format procedures and automatic analysis on a DNA Sequencer. The glycans of horseradish peroxidase with plant-specific core α(1,3)-fucose can be distinguished by the comparison of the glycan profiles obtained via PNGase A and F treatments. The peaks of the glycans with (91%) and without (1.2%) α(1,3)-fucose could be readily quantified and shown to harbor bisecting β(1,2)-xylose via simultaneous treatment with α(1,3)-mannosidase and β(1,2)-xylosidase. This optimized method was successfully applied to analyze N -glycans of plant-expressed recombinant antibody, which was engineered to contain a minor amount of glycan harboring β(1,2)-xylose. These results indicate that our DNA Sequencer-based method provides quantitative information for plant-specific N -glycan analysis in a high-throughput manner, which has not previously been achieved by glycan profiling based on mass spectrometry.
-
noninvasive diagnosis of liver cirrhosis using DNA Sequencer based total serum protein glycomics
Nature Medicine, 2004Co-Authors: Nico Callewaert, Wouter Laroy, Annelies Van Hecke, Joris Delanghe, Hans Van Vlierberghe, Roland ContrerasAbstract:We applied our 'clinical glycomics' technology, based on DNA Sequencer/fragment analyzers, to generate profiles of serum protein N-glycans of liver disease patients. This technology yielded a biomarker that distinguished compensated cirrhotic from noncirrhotic chronic liver disease patients, with 79% sensitivity and 86% specificity (100% sensitivity and specificity for decompensated cirrhosis). In combination with the clinical chemistry-based Fibrotest biomarker, compensated cirrhosis was detected with 100% specificity and 75% sensitivity. The current 'gold standard' for liver cirrhosis detection is an invasive, costly, often painful liver biopsy. Consequently, the highly specific set of biomarkers presented could obviate biopsy in many cirrhosis patients. This biomarker combination could eventually be used in follow-up examinations of chronic liver disease patients, to yield a warning that cirrhosis has developed and that the risk of complications (such as hepatocellular carcinoma) has increased considerably. Our clinical glycomics technique can easily be implemented in existing molecular diagnostics laboratories.
Yves Briers - One of the best experts on this subject based on the ideXlab platform.
-
Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria João Maurício Da Fonseca, Edita Jurak, Kim Kataja, A Van Landschoot, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
-
Analysis of the substrate specificity of -L-arabinofuranosidases by DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria Joao Mauricio Da Fonseca, Edita Jurak, Kim Kataja, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Anita Van Landschoot, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different -L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two -L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 -L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 -L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two -L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 -L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted -D-xylosyl residues, whereas a GH43 -L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
Minoru Asogawa - One of the best experts on this subject based on the ideXlab platform.
-
human short tandem repeat identification using a nanopore based DNA Sequencer a pilot study
Journal of Human Genetics, 2020Co-Authors: Minoru Asogawa, Ayumu Ohno, So Nakagawa, Eriko Ochiai, Yasuhiro Katahira, Megumi Sudo, Motoki Osawa, Masatoshi Sugisawa, Tadashi ImanishiAbstract:Short tandem repeats (STRs) are repetitive DNA sequences that are highly polymorphic and widely used for personal identification in the field of forensic medicine. The standard method for determining the repeat number of STRs is capillary electrophoresis of PCR products; however, the use of DNA sequencing has increased because it can identify same-sized alleles with nucleotide substitutions (iso-alleles). In this study, we performed human STR genotyping using a portable nanopore-based DNA Sequencer, the MinION, and evaluated its performance. Because the sequence quality obtained by MinION is considerably lower than those obtained with other DNA Sequencers, we developed an original scoring scheme for judging the genotypes from MinION reads. Analysis of seven human samples for 21–45 STR loci yielded an average of 857 thousand reads per sample, and the accuracy of genotyping and iso-allele identification reached 75.7% and 82%, respectively. Although the accuracy is higher than that reported previously, further improvements are required before this method can be practically applied.
Kim Kataja - One of the best experts on this subject based on the ideXlab platform.
-
Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria João Maurício Da Fonseca, Edita Jurak, Kim Kataja, A Van Landschoot, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
-
Analysis of the substrate specificity of -L-arabinofuranosidases by DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria Joao Mauricio Da Fonseca, Edita Jurak, Kim Kataja, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Anita Van Landschoot, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different -L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two -L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 -L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 -L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two -L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 -L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted -D-xylosyl residues, whereas a GH43 -L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
Tom Desmet - One of the best experts on this subject based on the ideXlab platform.
-
Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria João Maurício Da Fonseca, Edita Jurak, Kim Kataja, A Van Landschoot, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
-
Analysis of the substrate specificity of -L-arabinofuranosidases by DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria Joao Mauricio Da Fonseca, Edita Jurak, Kim Kataja, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Anita Van Landschoot, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA Sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different -L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two -L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 -L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 -L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two -L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 -L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted -D-xylosyl residues, whereas a GH43 -L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.