N-glycosylation has been defined as a prevalence protein post-translational modification involving in diverse physiological and pathological processes. N-glycosylation site occupancy is a physiological feature of glycoproteins and mainly contributes N-glycan macro-heterogeneity. The degree of self- N-glycosylation site occupancy has been reported to be correlated with the severity of the disease. Currently the technical obstacle has limited the progress in know-how of the regulation and function of the heterogeneity. Scientists confirmed that the characterization of the site-specific N-glycosylation including N-glycosylation site occupancy and site-specific glycan structure is vital for understanding of glycoprotein biosynthesis and function. Quantification of N-glycosylation site occupancy would reveal the critical role of macroheterogeneity in a variety of biological properties.
For analysis of the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to release the glycan from the protein. The formerly glycosylated asparagine undergoes deamidation and becomes an aspartic acid. The glycosylation site occupancy can be evaluated by comparing the abundance of the peptide containing aspartic acid to the one containing non-glycosylated asparagine. The general strategy of analysis of variable N-glycosylation site occupancy in glycoproteins comprise protein digestion, glycan release, glycopiptide separation by liquid chromatography and detection by mass spectrometry.
Figure 1. Workflow for Analysis of N-glycosylation site occupancy by LC-MS
There are also a couple of labeling or label free quantitative approaches to quantify N-glycosylation site occupancy by using LC-MS/MS.
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