41 research outputs found

    Top-Down Sequencing of O-Glycoproteins by In-Source Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Glycosylation Site Analysis

    No full text
    The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17–18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154

    Top-Down Sequencing of O-Glycoproteins by In-Source Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Glycosylation Site Analysis

    No full text
    The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17–18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154

    Top-Down Sequencing of O-Glycoproteins by In-Source Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Glycosylation Site Analysis

    No full text
    The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17–18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154

    Top-Down Sequencing of O-Glycoproteins by In-Source Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Glycosylation Site Analysis

    No full text
    The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17–18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154

    Top-Down Sequencing of O-Glycoproteins by In-Source Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Glycosylation Site Analysis

    No full text
    The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17–18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154

    Top-Down Sequencing of O-Glycoproteins by In-Source Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Glycosylation Site Analysis

    No full text
    The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17–18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154

    Top-Down Sequencing of O-Glycoproteins by In-Source Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Glycosylation Site Analysis

    No full text
    The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17–18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154

    Top-Down Sequencing of O-Glycoproteins by In-Source Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Glycosylation Site Analysis

    No full text
    The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17–18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154

    Top-Down Sequencing of O-Glycoproteins by In-Source Decay Matrix-Assisted Laser Desorption Ionization Mass Spectrometry for Glycosylation Site Analysis

    No full text
    The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17–18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154

    Differential Proteomics of Urinary Exovesicles from Classical Galactosemic Patients Reveals Subclinical Kidney Insufficiency

    No full text
    Classical galactosemia is caused by a nearly complete deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), resulting in a severely impaired galactose metabolism with galactose-1-phosphate and galactitol accumulation. Even on a galactose-restricted diet, patients develop serious long-term complications of the central nervous system and ovaries that may result from chronic cell-toxic effects exerted by endogenous galactose. To address the question of whether disease-associated cellular perturbations could affect the kidney function of the patients, we performed differential proteomics of detergent-resistant membranes from urinary exovesicles. Galactosemic samples (showing drastic shifts from high-mannose to complex-type N-glycosylation on exosomal N-glycoproteins) and healthy, sex-matched controls were analyzed in quadruplex iTRAQ experiments performed in biological and technical replicates. Particularly in the female patient group, the most striking finding was a drastic increase of abundant serum (glyco)­proteins, like albumin, leucine-rich α-2-glycoprotein, fetuin, immunoglobulins, prostaglandin H2 d-isomerase, and α-1-microglobulin protein (AMBP), pointing to a subclinical failure of kidney filter function in galactosemic patients and resulting in a heavy overload of exosomal membranes with adsorbed serum (glyco)­proteins. Several of these proteins are connected to TBMN and IgAN, proteinuria, and renal damage. The impairment of renal protein filtration was also indicated by increased protein contents derived from extracellular matrices and lysosomes
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