Targeted proteomics reveals quantitative differences in low abundance glycosyltransferases of patients with congenital disorders of glycosylation

Item Type:

Preprint

Title:

Creators Name:

Sakson, R. and Beedgen, L. and Bernhard, P. and Alp, K.M. and Lübbehusen, N. and Röth, R. and Niesler, B. and Mayer, M.P. and Thiel, C. and Ruppert, T.

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.

Source:

bioRxiv

Publisher:

Cold Spring Harbor Laboratory Press

Article Number:

2020.09.15.291732

Date:

16 September 2020

Official Publication:

https://doi.org/10.1101/2020.09.15.291732

Related to:

URL	URL Type
https://edoc.mdc-berlin.de/24048/	Final version

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