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Global serum glycoform profiling for the investigation of dystroglycanopathies & Congenital Disorders of Glycosylation.

Heywood WE, Bliss E, Mills P, Yuzugulen J, Carreno G, Clayton PT, Muntoni F, Worthington VC, Torelli S, Sebire NJ, Mills K, Grunewald S - Mol Genet Metab Rep (2016)

Bottom Line: These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases.In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies.The technique has further potential in monitoring patients for future treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: Centre for Inborn Errors of Metabolism, Great Ormond Street Hospital, Great Ormond Street, London WC1N 3JH, UK; Centre for Translational Omics, UCL Institute of Child Health & Great Ormond Street Hospital NHS Foundation Trust, London WC1N 1EH, UK.

ABSTRACT
The Congenital Disorders of Glycosylation (CDG) are an expanding group of genetic disorders which encompass a spectrum of glycosylation defects of protein and lipids, including N- & O-linked defects and among the latter are the muscular dystroglycanopathies (MD). Initial screening of CDG is usually based on the investigation of the glycoproteins transferrin, and/or apolipoprotein CIII. These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases. We describe a sensitive 2D-Differential Gel Electrophoresis (DIGE) method that provides a global analysis of the serum glycoproteome. Patient samples from PMM2-CDG (n = 5), CDG-II (n = 7), MD and known complex N- & O-linked glycosylation defects (n = 3) were analysed by 2D DIGE. Using this technique we demonstrated characteristic changes in mass and charge in PMM2-CDG and in charge in CDG-II for α1-antitrypsin, α1-antichymotrypsin, α2-HS-glycoprotein, ceruloplasmin, and α1-acid glycoproteins 1&2. Analysis of the samples with known N- & O-linked defects identified a lower molecular weight glycoform of C1-esterase inhibitor that was not observed in the N-linked glycosylation disorders indicating the change is likely due to affected O-glycosylation. In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies. The results demonstrate that the glycoform pattern is varied for some CDG patients not all glycoproteins are consistently affected and analysis of more than one protein in complex cases is warranted. 2D DIGE is an ideal method to investigate the global glycoproteome and is a potentially powerful tool and secondary test for aiding the complex diagnosis and sub classification of CDG. The technique has further potential in monitoring patients for future treatment strategies. In an era of shifting emphasis from gel- to mass-spectral based proteomics techniques, we demonstrate that 2D-DIGE remains a powerful method for studying global changes in post-translational modifications of proteins.

No MeSH data available.


Related in: MedlinePlus

Representative 15% 2D PAGE of albumin and IgG depleted serum. Highlighted proteins that had detectable changes in charge and mass in PMM2-CDG and CDG-II samples. The area defined by dashed lines equates to the optimised higher resolution area for glycoform analysis which was in the 3–5.6 pH range with a 10% acrylamide PAGE for proteins 40–200 kDa.
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f0005: Representative 15% 2D PAGE of albumin and IgG depleted serum. Highlighted proteins that had detectable changes in charge and mass in PMM2-CDG and CDG-II samples. The area defined by dashed lines equates to the optimised higher resolution area for glycoform analysis which was in the 3–5.6 pH range with a 10% acrylamide PAGE for proteins 40–200 kDa.

Mentions: The initial 2D DIGE experiment over the pH range 4–7 revealed clear changes in charge and mass for transferrin, α1-antitrypsin, α1-antichymotrypsin, α2-HS-glycoprotein and ceruloplasmin when patient samples were compared to that of the pooled control (Fig. 1).


Global serum glycoform profiling for the investigation of dystroglycanopathies & Congenital Disorders of Glycosylation.

Heywood WE, Bliss E, Mills P, Yuzugulen J, Carreno G, Clayton PT, Muntoni F, Worthington VC, Torelli S, Sebire NJ, Mills K, Grunewald S - Mol Genet Metab Rep (2016)

Representative 15% 2D PAGE of albumin and IgG depleted serum. Highlighted proteins that had detectable changes in charge and mass in PMM2-CDG and CDG-II samples. The area defined by dashed lines equates to the optimised higher resolution area for glycoform analysis which was in the 3–5.6 pH range with a 10% acrylamide PAGE for proteins 40–200 kDa.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834675&req=5

f0005: Representative 15% 2D PAGE of albumin and IgG depleted serum. Highlighted proteins that had detectable changes in charge and mass in PMM2-CDG and CDG-II samples. The area defined by dashed lines equates to the optimised higher resolution area for glycoform analysis which was in the 3–5.6 pH range with a 10% acrylamide PAGE for proteins 40–200 kDa.
Mentions: The initial 2D DIGE experiment over the pH range 4–7 revealed clear changes in charge and mass for transferrin, α1-antitrypsin, α1-antichymotrypsin, α2-HS-glycoprotein and ceruloplasmin when patient samples were compared to that of the pooled control (Fig. 1).

Bottom Line: These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases.In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies.The technique has further potential in monitoring patients for future treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: Centre for Inborn Errors of Metabolism, Great Ormond Street Hospital, Great Ormond Street, London WC1N 3JH, UK; Centre for Translational Omics, UCL Institute of Child Health & Great Ormond Street Hospital NHS Foundation Trust, London WC1N 1EH, UK.

ABSTRACT
The Congenital Disorders of Glycosylation (CDG) are an expanding group of genetic disorders which encompass a spectrum of glycosylation defects of protein and lipids, including N- & O-linked defects and among the latter are the muscular dystroglycanopathies (MD). Initial screening of CDG is usually based on the investigation of the glycoproteins transferrin, and/or apolipoprotein CIII. These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases. We describe a sensitive 2D-Differential Gel Electrophoresis (DIGE) method that provides a global analysis of the serum glycoproteome. Patient samples from PMM2-CDG (n = 5), CDG-II (n = 7), MD and known complex N- & O-linked glycosylation defects (n = 3) were analysed by 2D DIGE. Using this technique we demonstrated characteristic changes in mass and charge in PMM2-CDG and in charge in CDG-II for α1-antitrypsin, α1-antichymotrypsin, α2-HS-glycoprotein, ceruloplasmin, and α1-acid glycoproteins 1&2. Analysis of the samples with known N- & O-linked defects identified a lower molecular weight glycoform of C1-esterase inhibitor that was not observed in the N-linked glycosylation disorders indicating the change is likely due to affected O-glycosylation. In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies. The results demonstrate that the glycoform pattern is varied for some CDG patients not all glycoproteins are consistently affected and analysis of more than one protein in complex cases is warranted. 2D DIGE is an ideal method to investigate the global glycoproteome and is a potentially powerful tool and secondary test for aiding the complex diagnosis and sub classification of CDG. The technique has further potential in monitoring patients for future treatment strategies. In an era of shifting emphasis from gel- to mass-spectral based proteomics techniques, we demonstrate that 2D-DIGE remains a powerful method for studying global changes in post-translational modifications of proteins.

No MeSH data available.


Related in: MedlinePlus