The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Tamotsu Kanzaki - One of the best experts on this subject based on the ideXlab platform.
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Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases
2013Co-Authors: Befekadu Asfaw, Robert J. Desnick, Tamotsu Kanzaki, Jana Ledvinová, Robert Dobrovoln, Henk D. Bakker, Otto P. Van Diggelen, Jan G. N. De Jong, Amparo Chabas, Irene MaireAbstract:Abstract Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, �-N-acetylgalactosaminidase (�-NAGA) and �-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNA
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structural and immunocytochemical studies on α n acetylgalactosaminidase deficiency Schindler kanzaki disease
Journal of Human Genetics, 2004Co-Authors: Hitoshi Sakuraba, Tomoko Fukushige, Fumiko Matsuzawa, Sei-ichi Aikawa, Masaharu Kotani, Hiroshi Nakada, Hirofumi Doi, Tamotsu KanzakiAbstract:α-N-Acetylgalactosaminidase (α-NAGA) deficiency (Schindler/Kanzaki disease) is a clinically and pathologically heterogeneous genetic disease with a wide spectrum including an early onset neuroaxonal dystrophy (Schindler disease) and late onset angiokeratoma corporis diffusum (Kanzaki disease). In α-NAGA deficiency, there are discrepancies between the genotype and phenotype, and also between urinary excretion products (sialyl glycoconjugates) and a theoretical accumulated material (Tn-antigen; Gal NAcα1-Ο-Ser/Thr) resulting from a defect in α-NAGA. As for the former issue, previously reported genetic, biochemical and pathological data raise the question whether or not E325K mutation found in Schindler disease patients really leads to the severe phenotype of α-NAGA deficiency. The latter issue leads to the question of whether α-NAGA deficiency is associated with the basic pathogenesis of this disease. To clarify the pathogenesis of this disease, we performed structural and immunocytochemical studies. The structure of human α-NAGA deduced on homology modeling is composed of two domains, domain I, including the active site, and domain II. R329W/Q, identified in patients with Kanzaki disease have been deduced to cause drastic changes at the interface between domains I and II. The structural change caused by E325K found in patients with Schindler disease is localized on the N-terminal side of the tenth β-strand in domain II and is smaller than those caused by R329W/Q. Immunocytochemical analysis revealed that the main lysosomal accumulated material in cultured fibroblasts from patients with Kanzaki disease is Tn-antigen. These data suggest that a prototype of α-NAGA deficiency in Kanzaki disease and factors other than the defect of α-NAGA may contribute to severe neurological disorders, and Kanzaki disease is thought to be caused by a single enzyme deficiency.
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a new case of alpha n acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum with meniere s syndrome and without mental retardation
British Journal of Dermatology, 2001Co-Authors: Kazuo Kodama, Tomoko Fukushige, Shinichi Yotsumoto, Hitoshi Kobayashi, Akira Ohkawara, Noriko Yoshii, Yasuko Nagatsuka, Yoshio Hirabayashi, Tamotsu KanzakiAbstract:a-N-acetylgalactosaminidase (α-NAGA) deficiency is a rare hereditary lysosomal storage disease, and only three α-NAGA-deficient patients with angiokeratoma corporis diffusum (Kanzaki) have been described. We report a further case in a 47-year-old Japanese woman, the product of a consanguineous marriage. The remarkable findings in this patient were her normal intelligence, Meniere's syndrome, disturbance of peripheral sensory nerves, hearing loss and cardiac hypertrophy. α-NAGA enzyme activity in her plasma was 0.77% of the normal value. Other enzyme activities, such as α-galactosidase, β-galactosidase, α-L-fucosidase. β-mannosidase and aspartyl-glucosaminidase, were within normal limits. A large quantity of amino acid O-glycans was detected in her urine. Gene analysis revealed a novel point mutation (G→A transition) at nucleotide 11018 (986 in the cDNA) resulting in an Arg-329-Gln substitution. Kanzaki disease has the same enzyme defect as Schindler disease, but the manifestations are quite different.
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A new case of alpha-N-acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum, with Ménière's syndrome and without mental retardation.
The British journal of dermatology, 2001Co-Authors: Kazuo Kodama, Tomoko Fukushige, Shinichi Yotsumoto, Hitoshi Kobayashi, Riichiro Abe, Akira Ohkawara, Noriko Yoshii, Yasuko Nagatsuka, Yoshio Hirabayashi, Tamotsu KanzakiAbstract:alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency is a rare hereditary lysosomal storage disease, and only three alpha-NAGA-deficient patients with angiokeratoma corporis diffusum (Kanzaki) have been described. We report a further case in a 47-year-old Japanese woman, the product of a consanguineous marriage. The remarkable findings in this patient were her normal intelligence, Meniere's syndrome, disturbance of peripheral sensory nerves, hearing loss and cardiac hypertrophy. alpha-NAGA enzyme activity in her plasma was 0.77% of the normal value. Other enzyme activities, such as alpha-galactosidase, beta-galactosidase, alpha-L-fucosidase, beta-mannosidase and aspartylglucosaminidase, were within normal limits. A large quantity of amino acid O-glycans was detected in her urine. Gene analysis revealed a novel point mutation (G-->A transition) at nucleotide 11018 (986 in the cDNA) resulting in an Arg-329-Gln substitution. Kanzaki disease has the same enzyme defect as Schindler disease, but the manifestations are quite different.
Alina D. Zamfir - One of the best experts on this subject based on the ideXlab platform.
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Application of ion mobility tandem mass spectrometry to compositional and structural analysis of glycopeptides extracted from the urine of a patient diagnosed with Schindler disease
Rapid communications in mass spectrometry : RCM, 2015Co-Authors: Mirela Sarbu, Jasna Peter-katalinić, Feifei Zhu, David E. Clemmer, Alina D. ZamfirAbstract:Rationale Schindler disease is caused by the deficient activity of α-N-acetylgalactosaminidase, which leads to an abnormal accumulation of O-glycopeptides in tissues and body fluids. In this work the Schindler condition is for the first time approached by ion mobility (IMS) tandem mass spectrometry (MS/MS), for determining urine glycopeptide fingerprints and discriminate isomeric structures. Methods IMS-MS experiments were conducted on a Synapt G2s mass spectrometer operating in negative ion mode. A glycopeptide mixture extracted from the urine of a patient suffering from Schindler disease was dissolved in methanol and infused into the mass spectrometer by electrospray ionization using a syringe-pump system. MS/MS was performed by collision-induced dissociation (CID) at low energies, after mobility separation in the transfer cell. Data acquisition and processing were performed using MassLynx and Waters Driftscope software. Results IMS-MS data indicated that the attachment of one or two amino acids to the carbohydrate backbone has a minimal influence on the molecule conformation, which limits the discrimination of the free oligosaccharides from the glycosylated amino acids and dipeptides. The structural analysis by CID MS/MS in combination with IMS-MS of species exhibiting the same m/z but different configurations demonstrated for the first time the presence of positional isomers for some of the Schindler disease biomarker candidates. Conclusions The IMS-MS and CID MS/MS platform was for the first time optimized and applied to Schindler disease glycourinome. By this approach the separation and characterization of Neu5Ac positional isomers was possible. IMS CID MS/MS showed the ability to determine the type of the glycopeptide isomers from a series of possible candidates. Copyright © 2015 John Wiley & Sons, Ltd.
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Identification and structural characterization of novel O- and N-glycoforms in the urine of a Schindler disease patient by Orbitrap mass spectrometry.
Journal of mass spectrometry : JMS, 2015Co-Authors: Mirela Sarbu, Jasna Peter-katalinić, Cristian V. A. Munteanu, Liana Dehelean, Andrei J. Petrescu, Alina D. ZamfirAbstract:Schindler disease is an inherited metabolic disorder caused by the deficient activity of α-N-acetylgalactosaminidase enzyme. An accurate diagnosis requires, besides clinical examination, complex and costly biochemical and molecular genetic tests. In the last years, mass spectrometry (MS) based on nanofluidics and high-resolution instruments has become a successful alternative for disease diagnosis based on the investigation of O-glycopeptides in patient urine. A complex mixture of glycoforms extracted from the urine of a 3-year-old patient was investigated by Orbitrap MS equipped with Nanospray Flex Ion Source in the negative ion mode. For structural characterization of several molecular species, collision-induced dissociation MS2–MS3 was carried out using collision energy values within 20–60 eV range. By our approach, 39 novel species associated to this condition were identified, among which O-glycopeptides, free O-glycans and one structure corresponding to an N-glycan never characterized in the context of Schindler disease. The experiments conducted at a resolution of 60 000 allowed the discrimination and identification of a total number of 69 different species with an average mass accuracy of 9.87 ppm, an in-run reproducibility of almost 100%, an experiment-to-experiment and day-to-day reproducibility of about 95%. This study brings contributions in the diagnosis of Schindler disease through the elucidation of potential biomarker species in urine. Our multistage MS results completed with 39 new glycoforms the inventory of potential biomarker structures associated to Schindler disease. For the first time, an N-glycan was identified and structurally characterized in Schindler patient urine, which opens new research directions in the field. Copyright © 2015 John Wiley & Sons, Ltd.
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Automated chip-nanoelectrospray mass spectrometry for glycourinomics in Schindler disease type I
Carbohydrate research, 2014Co-Authors: Mirela Sarbu, Adrian C. Robu, Jasna Peter-katalinić, Alina D. ZamfirAbstract:In this study an integrative mass spectrometry (MS) approach based on fully automated chip-nanoelectrospray quadrupole time-of-flight was optimized and applied for the discovery and structural characterization of O-glycopeptides in a fraction from the urine of a patient diagnosed with Schindler disease type I. A mixture of O-glycopeptides extracted and purified from an age matched healthy subject served as the control. 49 glycoforms were discovered in the investigated urine fraction from Schindler disease versus only 14 in control urine. Structures with relevant biological significance, previously not described, such as O-fucosylated tetrasaccharides and chains up to pentadecamers O-linked to serine, threonine, or threonine-proline were identified in the pathological urine and characterized by tandem MS (MS/MS). A number of 29 species discovered here, most of which with long chain glycans, were not previously reported as associated to this condition. All glycopeptides were detected in only 1 min analysis time, with a sample consumption situated in the femtomole range.
Scott C. Garman - One of the best experts on this subject based on the ideXlab platform.
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Pharmacological chaperones for human α-N-acetylgalactosaminidase
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Nathaniel E. Clark, Matthew C. Metcalf, Daniel Best, George W. J. Fleet, Scott C. GarmanAbstract:Schindler/Kanzaki disease is an inherited metabolic disease with no current treatment options. This neurologic disease results from a defect in the lysosomal α-N-acetylgalactosaminidase (α-NAGAL) enzyme. In this report, we show evidence that the iminosugar DGJNAc can inhibit, stabilize, and chaperone human α-NAGAL both in vitro and in vivo. We demonstrate that a related iminosugar DGJ (currently in phase III clinical trials for another metabolic disorder, Fabry disease) can also chaperone human α-NAGAL in Schindler/Kanzaki disease. The 1.4- and 1.5-A crystal structures of human α-NAGAL complexes reveal the different binding modes of iminosugars compared with glycosides. We show how differences in two functional groups result in >9 kcal/mol of additional binding energy and explain the molecular interactions responsible for the unexpectedly high affinity of the pharmacological chaperones. These results open two avenues for treatment of Schindler/Kanzaki disease and elucidate the atomic basis for pharmacological chaperoning in the entire family of lysosomal storage diseases.
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Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases
The Journal of biological chemistry, 2010Co-Authors: Ivan B. Tomasic, Nathaniel E. Clark, Matthew Metcalf, Abigail I. Guce, Scott C. GarmanAbstract:The human lysosomal enzymes α-galactosidase (α-GAL, EC 3.2.1.22) and α-N-acetylgalactosaminidase (α-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of α- GAL and α-NAGAL. The engineered α-GAL (which we call α-GALSA) retains the antigenicity of α-GAL but has acquired the enzymatic specificity of α-NAGAL. Conversely, the engineered α-NAGAL (which we call α-NAGALEL) retains the antigenicity of α-NAGAL but has acquired the enzymatic specificity of the α-GAL enzyme. Comparison of the crystal structures of the designed enzyme α-GALSA to the wild-type enzymes shows that active sites of α-GALSA and α-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.
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The 1.9 Å structure of human α-N-acetylgalactosaminidase: The molecular basis of Schindler and Kanzaki diseases
Journal of molecular biology, 2009Co-Authors: Nathaniel E. Clark, Scott C. GarmanAbstract:α-N-acetylgalactosaminidase (α-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal α-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of α-NAGAL activity results in the lysosomal storage disorders Schindler disease and Kanzaki disease. To better understand the molecular defects in the diseases, we determined the crystal structure of human α-NAGAL after expressing wild-type and glycosylation-deficient glycoproteins in recombinant insect cell expression systems. We measured the enzymatic parameters of our purified wild-type and mutant enzymes, establishing their enzymatic equivalence. To investigate the binding specificity and catalytic mechanism of the human α-NAGAL enzyme, we determined three crystallographic complexes with different catalytic products bound in the active site of the enzyme. To better understand how individual defects in the α-NAGAL glycoprotein lead to Schindler disease, we analyzed the effect of disease-causing mutations on the three-dimensional structure.
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Structural studies on α-GAL and α-NAGAL : The atomic basis of Fabry and Schindler diseases
Biocatalysis and Biotransformation, 2006Co-Authors: Scott C. GarmanAbstract:Until 2002, no representative structures existed for the glycoside hydrolase family 27 and clan D, which include the enzymes α-galactosidase (α-GAL, E.C. 3.2.1.22) and α-N-acetylgalactosaminidase (α-NAGAL, E.C. 3.2.1.49). Since then, four structures have been determined: α-NAGAL from chicken and α-GAL from rice, human, and Trichoderma reesei. This manuscript addresses the structure and function of the family 27 glycoside hydrolases, including the mechanism and substrate specificity. In humans, mutations in the α-GAL and α-NAGAL enzymes lead to the lysosomal storage disorders Fabry disease and Schindler disease, respectively, which are characterized by the incomplete degradation of carbohydrates with terminal α-galactose and α-N-acetylgalactosamine, respectively. The hundreds of mutations identified in Fabry and Schindler disease patients will be addressed in the light of the structures, revealing the atomic basis for the diseases.
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the 1 9 a structure of α n acetylgalactosaminidase molecular basis of glycosidase deficiency diseases
Structure, 2002Co-Authors: Scott C. Garman, Linda I Hannick, David N GarbocziAbstract:Abstract In the lysosome, glycosidases degrade glycolipids, glycoproteins, and oligosaccharides. Mutations in glycosidases cause disorders characterized by the deposition of undegraded carbohydrates. Schindler and Fabry diseases are caused by the incomplete degradation of carbohydrates with terminal α-N-acetylgalactosamine and α-galactose, respectively. Here we present the X-ray structure of α-N-acetylgalactosaminidase (α-NAGAL), the glycosidase that removes α-N-acetylgalactosamine, and the structure with bound ligand. The active site residues of α-NAGAL are conserved in the closely related enzyme α-galactosidase A (α-GAL). The structure demonstrates the catalytic mechanisms of both enzymes and reveals the structural basis of mutations causing Schindler and Fabry diseases. As α-NAGAL and α-GAL produce type O "universal donor" blood from type A and type B blood, the α-NAGAL structure will aid in the engineering of improved enzymes for blood conversion.
Tomoko Fukushige - One of the best experts on this subject based on the ideXlab platform.
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Improved Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometric Method for Identifi- cation of Amino Acid O-Glycosides in Patients with -N-Acetylgalactosaminidase Deficiency, Masahisa Ho-
2015Co-Authors: Tomoko Fukushige, Takeyori SahekiAbstract:u.ac.jp) Thin-layer chromatography (TLC) has been widely ap-plied to the diagnosis of lysosomal storage diseases by analyzing abnormally excreted oligosaccharides in urine. However, TLC does not necessarily identify the excreted compounds accurately. Labeling the oligosaccharides with a tag, such as 8-aminonaphthalene-1,3,6-trisulfonic acid, allows easy identification of the excreted com-pounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (1). However, this labeling is based on reductive amination of the reductive end of the oligosaccharides with the labeling compound, so abnormal amino acid O-glycosides with no reducing end cannot be identified. Indeed, patients with Kanzaki/Schindler disease or aspartylglucosaminuria
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structural and immunocytochemical studies on α n acetylgalactosaminidase deficiency Schindler kanzaki disease
Journal of Human Genetics, 2004Co-Authors: Hitoshi Sakuraba, Tomoko Fukushige, Fumiko Matsuzawa, Sei-ichi Aikawa, Masaharu Kotani, Hiroshi Nakada, Hirofumi Doi, Tamotsu KanzakiAbstract:α-N-Acetylgalactosaminidase (α-NAGA) deficiency (Schindler/Kanzaki disease) is a clinically and pathologically heterogeneous genetic disease with a wide spectrum including an early onset neuroaxonal dystrophy (Schindler disease) and late onset angiokeratoma corporis diffusum (Kanzaki disease). In α-NAGA deficiency, there are discrepancies between the genotype and phenotype, and also between urinary excretion products (sialyl glycoconjugates) and a theoretical accumulated material (Tn-antigen; Gal NAcα1-Ο-Ser/Thr) resulting from a defect in α-NAGA. As for the former issue, previously reported genetic, biochemical and pathological data raise the question whether or not E325K mutation found in Schindler disease patients really leads to the severe phenotype of α-NAGA deficiency. The latter issue leads to the question of whether α-NAGA deficiency is associated with the basic pathogenesis of this disease. To clarify the pathogenesis of this disease, we performed structural and immunocytochemical studies. The structure of human α-NAGA deduced on homology modeling is composed of two domains, domain I, including the active site, and domain II. R329W/Q, identified in patients with Kanzaki disease have been deduced to cause drastic changes at the interface between domains I and II. The structural change caused by E325K found in patients with Schindler disease is localized on the N-terminal side of the tenth β-strand in domain II and is smaller than those caused by R329W/Q. Immunocytochemical analysis revealed that the main lysosomal accumulated material in cultured fibroblasts from patients with Kanzaki disease is Tn-antigen. These data suggest that a prototype of α-NAGA deficiency in Kanzaki disease and factors other than the defect of α-NAGA may contribute to severe neurological disorders, and Kanzaki disease is thought to be caused by a single enzyme deficiency.
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a new case of alpha n acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum with meniere s syndrome and without mental retardation
British Journal of Dermatology, 2001Co-Authors: Kazuo Kodama, Tomoko Fukushige, Shinichi Yotsumoto, Hitoshi Kobayashi, Akira Ohkawara, Noriko Yoshii, Yasuko Nagatsuka, Yoshio Hirabayashi, Tamotsu KanzakiAbstract:a-N-acetylgalactosaminidase (α-NAGA) deficiency is a rare hereditary lysosomal storage disease, and only three α-NAGA-deficient patients with angiokeratoma corporis diffusum (Kanzaki) have been described. We report a further case in a 47-year-old Japanese woman, the product of a consanguineous marriage. The remarkable findings in this patient were her normal intelligence, Meniere's syndrome, disturbance of peripheral sensory nerves, hearing loss and cardiac hypertrophy. α-NAGA enzyme activity in her plasma was 0.77% of the normal value. Other enzyme activities, such as α-galactosidase, β-galactosidase, α-L-fucosidase. β-mannosidase and aspartyl-glucosaminidase, were within normal limits. A large quantity of amino acid O-glycans was detected in her urine. Gene analysis revealed a novel point mutation (G→A transition) at nucleotide 11018 (986 in the cDNA) resulting in an Arg-329-Gln substitution. Kanzaki disease has the same enzyme defect as Schindler disease, but the manifestations are quite different.
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A new case of alpha-N-acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum, with Ménière's syndrome and without mental retardation.
The British journal of dermatology, 2001Co-Authors: Kazuo Kodama, Tomoko Fukushige, Shinichi Yotsumoto, Hitoshi Kobayashi, Riichiro Abe, Akira Ohkawara, Noriko Yoshii, Yasuko Nagatsuka, Yoshio Hirabayashi, Tamotsu KanzakiAbstract:alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency is a rare hereditary lysosomal storage disease, and only three alpha-NAGA-deficient patients with angiokeratoma corporis diffusum (Kanzaki) have been described. We report a further case in a 47-year-old Japanese woman, the product of a consanguineous marriage. The remarkable findings in this patient were her normal intelligence, Meniere's syndrome, disturbance of peripheral sensory nerves, hearing loss and cardiac hypertrophy. alpha-NAGA enzyme activity in her plasma was 0.77% of the normal value. Other enzyme activities, such as alpha-galactosidase, beta-galactosidase, alpha-L-fucosidase, beta-mannosidase and aspartylglucosaminidase, were within normal limits. A large quantity of amino acid O-glycans was detected in her urine. Gene analysis revealed a novel point mutation (G-->A transition) at nucleotide 11018 (986 in the cDNA) resulting in an Arg-329-Gln substitution. Kanzaki disease has the same enzyme defect as Schindler disease, but the manifestations are quite different.
Mirela Sarbu - One of the best experts on this subject based on the ideXlab platform.
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Application of ion mobility tandem mass spectrometry to compositional and structural analysis of glycopeptides extracted from the urine of a patient diagnosed with Schindler disease
Rapid communications in mass spectrometry : RCM, 2015Co-Authors: Mirela Sarbu, Jasna Peter-katalinić, Feifei Zhu, David E. Clemmer, Alina D. ZamfirAbstract:Rationale Schindler disease is caused by the deficient activity of α-N-acetylgalactosaminidase, which leads to an abnormal accumulation of O-glycopeptides in tissues and body fluids. In this work the Schindler condition is for the first time approached by ion mobility (IMS) tandem mass spectrometry (MS/MS), for determining urine glycopeptide fingerprints and discriminate isomeric structures. Methods IMS-MS experiments were conducted on a Synapt G2s mass spectrometer operating in negative ion mode. A glycopeptide mixture extracted from the urine of a patient suffering from Schindler disease was dissolved in methanol and infused into the mass spectrometer by electrospray ionization using a syringe-pump system. MS/MS was performed by collision-induced dissociation (CID) at low energies, after mobility separation in the transfer cell. Data acquisition and processing were performed using MassLynx and Waters Driftscope software. Results IMS-MS data indicated that the attachment of one or two amino acids to the carbohydrate backbone has a minimal influence on the molecule conformation, which limits the discrimination of the free oligosaccharides from the glycosylated amino acids and dipeptides. The structural analysis by CID MS/MS in combination with IMS-MS of species exhibiting the same m/z but different configurations demonstrated for the first time the presence of positional isomers for some of the Schindler disease biomarker candidates. Conclusions The IMS-MS and CID MS/MS platform was for the first time optimized and applied to Schindler disease glycourinome. By this approach the separation and characterization of Neu5Ac positional isomers was possible. IMS CID MS/MS showed the ability to determine the type of the glycopeptide isomers from a series of possible candidates. Copyright © 2015 John Wiley & Sons, Ltd.
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Identification and structural characterization of novel O- and N-glycoforms in the urine of a Schindler disease patient by Orbitrap mass spectrometry.
Journal of mass spectrometry : JMS, 2015Co-Authors: Mirela Sarbu, Jasna Peter-katalinić, Cristian V. A. Munteanu, Liana Dehelean, Andrei J. Petrescu, Alina D. ZamfirAbstract:Schindler disease is an inherited metabolic disorder caused by the deficient activity of α-N-acetylgalactosaminidase enzyme. An accurate diagnosis requires, besides clinical examination, complex and costly biochemical and molecular genetic tests. In the last years, mass spectrometry (MS) based on nanofluidics and high-resolution instruments has become a successful alternative for disease diagnosis based on the investigation of O-glycopeptides in patient urine. A complex mixture of glycoforms extracted from the urine of a 3-year-old patient was investigated by Orbitrap MS equipped with Nanospray Flex Ion Source in the negative ion mode. For structural characterization of several molecular species, collision-induced dissociation MS2–MS3 was carried out using collision energy values within 20–60 eV range. By our approach, 39 novel species associated to this condition were identified, among which O-glycopeptides, free O-glycans and one structure corresponding to an N-glycan never characterized in the context of Schindler disease. The experiments conducted at a resolution of 60 000 allowed the discrimination and identification of a total number of 69 different species with an average mass accuracy of 9.87 ppm, an in-run reproducibility of almost 100%, an experiment-to-experiment and day-to-day reproducibility of about 95%. This study brings contributions in the diagnosis of Schindler disease through the elucidation of potential biomarker species in urine. Our multistage MS results completed with 39 new glycoforms the inventory of potential biomarker structures associated to Schindler disease. For the first time, an N-glycan was identified and structurally characterized in Schindler patient urine, which opens new research directions in the field. Copyright © 2015 John Wiley & Sons, Ltd.
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Automated chip-nanoelectrospray mass spectrometry for glycourinomics in Schindler disease type I
Carbohydrate research, 2014Co-Authors: Mirela Sarbu, Adrian C. Robu, Jasna Peter-katalinić, Alina D. ZamfirAbstract:In this study an integrative mass spectrometry (MS) approach based on fully automated chip-nanoelectrospray quadrupole time-of-flight was optimized and applied for the discovery and structural characterization of O-glycopeptides in a fraction from the urine of a patient diagnosed with Schindler disease type I. A mixture of O-glycopeptides extracted and purified from an age matched healthy subject served as the control. 49 glycoforms were discovered in the investigated urine fraction from Schindler disease versus only 14 in control urine. Structures with relevant biological significance, previously not described, such as O-fucosylated tetrasaccharides and chains up to pentadecamers O-linked to serine, threonine, or threonine-proline were identified in the pathological urine and characterized by tandem MS (MS/MS). A number of 29 species discovered here, most of which with long chain glycans, were not previously reported as associated to this condition. All glycopeptides were detected in only 1 min analysis time, with a sample consumption situated in the femtomole range.
