Quantitative investigation of human cell surface N-glycoprotein dynamics.

Surface glycoproteins regulate nearly every extracellular event and they are dynamic for cells to adapt to the ever-changing extracellular environment. These glycoproteins contain a wealth of information on cellular development and disease states, and have significant biomedical implications. Systematic investigation of surface glycoproteins will result in a better understanding of surface protein functions, cellular activities and the molecular mechanisms of disease. However, it is extraordinarily challenging to specifically and globally analyze surface glycoproteins. Here we designed the first method to systematically analyze surface glycoprotein dynamics and measure their half-lives by integrating pulse-chase labeling, selective enrichment of surface glycoproteins, and multiplexed proteomics. The current results clearly demonstrated that surface glycoproteins with catalytic activities were more stable than those with binding and receptor activities. Glycosylation sites located outside of any domain had a notably longer median half-life than those within domains, which strongly suggests that glycans within domains regulate protein interactions with other molecules while those outside of domains mainly play a role in protecting the protein from degradation. This method can be extensively applied to biological and biomedical research.