Dissecting the subcellular secretory glycoproteome with mass spectrometry proteomics

Zacchi LF1, Phung T2 and Schulz BL1,2

  1. Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland.
  2. School of Chemistry and Molecular Biosciences, The University of Queensland.

N-glycosylation is a critical post-translational modification that influences the folding and function of 1/3rd of the cellular proteome. The biosynthesis of N-glycoproteins begins in the endoplasmic reticulum (ER), where an oligosaccharide is transferred to selected asparagine residues in nascent polypeptides by the enzyme oligosaccharyltransferase. The presence of N-glycans at specific sites is critical for productive protein folding in the ER, and defects in this process perturb glycoprotein folding, secretion, and function at a systems level. We have developed integrated subcellular fractionation and SWATH glycoproteomic workflows to understand the causes and consequences of changes in the N-glycosylation biosynthetic pathway. We combined biochemical subcellular fractionation methods with quantitative SWATH-MS glycoproteomic and proteomic workflows to measure the response to a range of genetic and chemical perturbations to N-glycoprotein biosynthesis. We optimized biochemical fractionation methods in yeast to enable precise analysis of the subcellular proteome and glycoproteome. This enabled quantitative measurement of subcellular proteomes and site-specific and global profiling of glycan occupancy and structure. We tested these methods in yeast with defined defects in N-glycosylation, and then expanded our analysis to profile the quantitative effects of combined defects in glycoprotein biosynthesis and protein quality control on glycoprotein maturation. Our results give key insights into the effect of site-specific glycosylation on glycoprotein quality control processes, and our methods will be useful in diverse applications in industrial and medical glycobiotechnology.