Surface Glycoproteomic Analysis Reveals That Both Unique and Differential Expression of Surface Glycoproteins Determine the Cell Type

Proteins
on the cell surface are frequently glycosylated, and they
are essential for cells. Surface glycoproteins regulate nearly every
extracellular event, but compared with global analysis of proteins,
comprehensive and site-specific analysis of surface glycoproteins
is much more challenging and dramatically understudied. Here, combining
metabolic labeling, click-chemistry and enzymatic reactions, and mass
spectrometry-based proteomics, we globally characterized surface glycoproteins
from eight popular types of human cells. This integrative and effective
method allowed for the identification of 2172 N-glycosylation sites
and 1047 surface glycoproteins. The distribution and occurrence of
N-glycosylation sites were systematically investigated, and protein
secondary structures were found to have a dramatic influence on glycosylation
sites. As expected, most sites are located on disordered regions.
For the sites with the motif N-!P-C, about one-third of them are located on helix structures, while
those with the motif N-!P-S/T prefer strand structures. There is almost
no correlation between the number of glycosylation sites and protein
length, but the number of sites corresponds well with the frequencies
of the motif. Quantification results reveal that besides cell-specific
glycoproteins, the uniqueness of each cell type further arises from
differential expression of surface glycoproteins. The current research
indicates that multiple surface glycoproteins including their abundances
need to be considered for cell classification rather than a single
cluster of differentiation (CD) protein normally used in conventional
methods. These results provide valuable information to the glycoscience
and biomedical communities and aid in the discovery of surface glycoproteins
as disease biomarkers and drug targets.