Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.15.291732v1?rss=1 Authors: Sakson, R., Beedgen, L., Bernhard, P., Alp, K. M., Luebbehusen, N., Roeth, R., Niesler, B., Mayer, M. P., Thiel, C., Ruppert, T. Abstract: Protein glycosylation is essential in all domains of life and its impairment can result in severe human 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. This HEK 293T cells-derived standard could be used to reliably quantify 22 glycosyltransferases in HeLa cells and skin fibroblast cell lines. In addition, we showed that the MRM assay is easily transferable between laboratories. We then analyzed fibroblasts derived from CDG type I patients with defects 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 ALG proteins not directly affected by a genetic defect 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. Copy rights belong to original authors. Visit the link for more info