pr5003005_si_002.pdf (40.24 kB)
Rapid Analysis of Cell Surface N‑Glycosylation from Living Cells Using Mass Spectrometry
journal contribution
posted on 2014-12-05, 00:00 authored by Houda Hamouda, Matthias Kaup, Mujib Ullah, Markus Berger, Volker Sandig, Rudolf Tauber, Véronique BlanchardCell
surfaces are covered with a dense carbohydrate layer referred
to as the glycocalyx. Because different cell types express different
glycan signatures, it is of paramount importance to have robust methods
to analyze the glycome of living cells. To achieve this, a common
procedure involves cell lysis and extraction of membrane (glyco)proteins
and yields a major proportion of high-mannose N-glycans that most
likely stem from intracellular proteins derived from the ER. Using
HEK 293 cells as a model system, we developed a reproducible, sensitive,
and fast method to profile surface N-glycosylation from living cells.
We directly released glycopeptides from cell surfaces through tryptic
digestion of freshly harvested and vital cells, thereby improving
the detection and quantification of complex-type N-glycans by increasing
their relative amount from 14 to 85%. It was also possible to detect
25 additional structures in HEK 293, 48 in AGE1.HN, 42 in CHO-K1,
and 51 in Hep G2 cells. The additional signals provided deeper insight
into cell-type-specific N-glycan features such as antennarity, fucosylation,
and sialylation. Thus, this protocol, which can potentially be applied
to any cells, will be useful in the fields of glycobiotechnology and
biomarker discovery.