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

Abstract

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 *

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

“…Analyzing glycoproteomes from whole-cell lysate generates confounding artifacts that complicate determination of what glycoforms are biologically relevant and present on the cell surface. Several approaches have been developed to label cell-surface glycoproteins on live cells, prior to cell lysis and protein extraction ( 306 , 307 ). One variation functions similarly to the approaches above: sialic acids are oxidized on live cells via mild periodate treatment, and aldehyde-containing sialylated glycopeptides are captured on hydrazide beads.…”

Advances in mass spectrometry‐based glycoproteomics: An update covering the period 2017–2021

“…Witzke and co-workers [76] compared two labeling compounds, DBCO-sulfo-biotin and DBCO-PEG4desthiobiotin, and found that 356 proteins were quantified using the biotin probe and 463 using the desthiobiotin probe. Wu and coworkers [77,78] combined azido-based metabolic labeling and copper-free click chemistry with MS-based proteomics to selectively enrich cell surface glycoproteins and reveal the differential expression of surface glycoproteins of different cell types. Recently, they combined a boosting approach that incorporated TMT with Ac4GalNAz labeling and copper-free chemistry to label secreted glycoproteins from cultured cells without serum starvation [79].…”

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“…2,9 The majority of cell surface proteins are glycosylated, and the most common ones are protein N-and Oglycosylation. [10][11][12][13][14] Aberrant glycosylation can alter proteinprotein interactions, which has been correlated with disease initiation and development. 15,16 For instance, changes in the glycosylation of CD44, a cell surface receptor involved in cancer proliferation and migration, can signicantly affect its binding towards the ligand of hyaluronic acid and thus change cancer cell signaling.…”