Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins

Although the classification of cell types often relies on the identification of cell surface proteins as differentiation markers, flow cytometry requires suitable antibodies and currently permits detection of only up to a dozen differentiation markers in a single measurement. We use multiplexed mass-spectrometric identification of several hundred N-linked glycosylation sites specifically from cell surface-exposed glycoproteins to phenotype cells without antibodies in an unbiased fashion and without a priori knowledge. We apply our cell surface-capturing (CSC) technology, which covalently labels extracellular glycan moieties on live cells, to the detection and relative quantitative comparison of the cell surface N-glycoproteomes of T and B cells, as well as to monitor changes in the abundance of cell surface N-glycoprotein markers during T-cell activation and the controlled differentiation of embryonic stem cells into the neural lineage. A snapshot view of the cell surface N-glycoproteins will enable detection of panels of N-glycoproteins as potential differentiation markers that are currently not accessible by other means.