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Targeted Proteomics Reveals Quantitative Differences in Low Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation

View ORCID ProfileRoman Sakson, View ORCID ProfileLars Beedgen, View ORCID ProfilePatrick Bernhard, View ORCID ProfileKeziban M. Alp, Nicole Lübbehusen, Ralph Röth, Beate Niesler, View ORCID ProfileMatthias P. Mayer, View ORCID ProfileChristian Thiel, Thomas Ruppert
doi: https://doi.org/10.1101/2020.09.15.291732
Roman Sakson
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
2HBIGS, Heidelberg Biosciences International Graduate School, Heidelberg University, 69120 Heidelberg, Germany
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Lars Beedgen
3Center for Child and Adolescent Medicine, Department Pediatrics I, Heidelberg University, 69120 Heidelberg, Germany
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Patrick Bernhard
4Institute for Surgical Pathology, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
5Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Germany
6Faculty of Biology, Albert-Ludwigs-University Freiburg, Freiburg, Germany
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Keziban M. Alp
7Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 12135 Berlin, Germany
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Nicole Lübbehusen
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Ralph Röth
8Department of Human Molecular Genetics, University Hospital Heidelberg, Heidelberg, Germany
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Beate Niesler
8Department of Human Molecular Genetics, University Hospital Heidelberg, Heidelberg, Germany
9Interdisciplinary Center for Neurosciences, Heidelberg University, Heidelberg, Germany
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Matthias P. Mayer
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Christian Thiel
3Center for Child and Adolescent Medicine, Department Pediatrics I, Heidelberg University, 69120 Heidelberg, Germany
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Thomas Ruppert
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
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  • For correspondence: t.ruppert@zmbh.uni-heidelberg.de
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Abstract

Protein glycosylation is essential in all domains of life and its mutational impairment in humans can result in severe diseases named Congenital Disorders of Glycosylation (CDGs). Studies on molecular level are however challenging, because many glycosyltransferases in the endoplasmic reticulum (ER) are low abundance membrane proteins. We established a comprehensive multiple reaction monitoring (MRM) assay to quantify most human glycosyltransferases involved in the processes of N-glycosylation,O- and C-mannosylation in the ER. To increase reproducibility, a membrane protein fraction of isotopically labeled HEK 293T cells was used as an internal standard. With this internal standard the MRM assay is easily transferable between laboratories. 22 glycosyltransferases could be reliably quantified from whole cell lysates of HEK 293T cells, HeLa cells and skin fibroblast cell lines. We then analyzed fibroblasts derived from CDG type I patients with mutations in the ALG1,ALG2 or ALG11 gene. Mutations in ALG1 or ALG2 gene strongly reduced the levels of the ALG1 and ALG2 protein, respectively. In contrast, the levels of all other glycosyltransferases remained unchanged, which was unexpected given evidence that the ALG1, ALG2 and ALG11 proteins form a stable complex. This study describes an efficient workflow for the development of MRM assays for low abundance proteins, establishes a ready-to-use tool for the comprehensive quantification of ER-localized glycosyltransferases and provides new insight into the organization of disease-relevant glycosylation processes.

Competing Interest Statement

The authors have declared no competing interest.

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Posted September 16, 2020.
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Targeted Proteomics Reveals Quantitative Differences in Low Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation
Roman Sakson, Lars Beedgen, Patrick Bernhard, Keziban M. Alp, Nicole Lübbehusen, Ralph Röth, Beate Niesler, Matthias P. Mayer, Christian Thiel, Thomas Ruppert
bioRxiv 2020.09.15.291732; doi: https://doi.org/10.1101/2020.09.15.291732
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Targeted Proteomics Reveals Quantitative Differences in Low Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation
Roman Sakson, Lars Beedgen, Patrick Bernhard, Keziban M. Alp, Nicole Lübbehusen, Ralph Röth, Beate Niesler, Matthias P. Mayer, Christian Thiel, Thomas Ruppert
bioRxiv 2020.09.15.291732; doi: https://doi.org/10.1101/2020.09.15.291732

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