Enzymatic Tagging of Glycoproteins on the Cell Surface for Their Global and Site-Specific Analysis with Mass Spectrometry

The
cell surface is normally covered with sugars that are bound
to lipids or proteins. Surface glycoproteins play critically important
roles in many cellular events, including cell–cell communications,
cell–matrix interactions, and response to environmental cues.
Aberrant protein glycosylation on the cell surface is often a hallmark
of human diseases such as cancer and infectious diseases. Global analysis
of surface glycoproteins will result in a better understanding of
glycoprotein functions and the molecular mechanisms of diseases and
the discovery of surface glycoproteins as biomarkers and drug targets.
Here, an enzyme is exploited to tag surface glycoproteins, generating
a chemical handle for their selective enrichment prior to mass spectrometric
(MS) analysis. The enzymatic reaction is very efficient, and the reaction
conditions are mild, which are well-suited for surface glycoprotein
tagging. For biologically triplicate experiments, on average 953 N-glycosylation
sites on 393 surface glycoproteins per experiment were identified
in MCF7 cells. Integrating chemical and enzymatic reactions with MS-based
proteomics, the current method is highly effective to globally and
site-specifically analyze glycoproteins only located on the cell surface.
Considering the importance of surface glycoproteins, this method is
expected to have extensive applications to advance glycoscience.