Click Chemistry-mediated Biotinylation Reveals a Function for the Protease BACE1 in Modulating the Neuronal Surface Glycoproteome

Mol Cell Proteomics. 2018 Aug;17(8):1487-1501. doi: 10.1074/mcp.RA118.000608. Epub 2018 May 1.


The cell surface proteome is dynamic and has fundamental roles in cell signaling. Many surface membrane proteins are proteolytically released into a cell's secretome, where they can have additional functions in cell-cell-communication. Yet, it remains challenging to determine the surface proteome and to compare it to the cell secretome, under serum-containing cell culture conditions. Here, we set up and evaluated the 'surface-spanning protein enrichment with click sugars' (SUSPECS) method for cell surface membrane glycoprotein biotinylation, enrichment and label-free quantitative mass spectrometry. SUSPECS is based on click chemistry-mediated labeling of glycoproteins, is compatible with labeling of living cells and can be combined with secretome analyses in the same experiment. Immunofluorescence-based confocal microscopy demonstrated that SUSPECS selectively labeled cell surface proteins. Nearly 700 transmembrane glycoproteins were consistently identified at the surface of primary neurons. To demonstrate the utility of SUSPECS, we applied it to the protease BACE1, which is a key drug target in Alzheimer's disease. Pharmacological BACE1-inhibition selectively remodeled the neuronal surface glycoproteome, resulting in up to 7-fold increased abundance of the BACE1 substrates APP, APLP1, SEZ6, SEZ6L, CNTN2, and CHL1, whereas other substrates were not or only mildly affected. Interestingly, protein changes at the cell surface only partly correlated with changes in the secretome. Several altered proteins were validated by immunoblots in neurons and mouse brains. Apparent nonsubstrates, such as TSPAN6, were also increased, indicating that BACE1-inhibition may lead to unexpected secondary effects. In summary, SUSPECS is broadly useful for determination of the surface glycoproteome and its correlation with the secretome.

Keywords: Alzheimer's disease; BACE1; Cell surface; Click chemistry; Label-free quantification; Mass Spectrometry; N-glycosylation; cell surface display technologies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid Precursor Protein Secretases / antagonists & inhibitors
  • Amyloid Precursor Protein Secretases / metabolism*
  • Animals
  • Aspartic Acid Endopeptidases / antagonists & inhibitors
  • Aspartic Acid Endopeptidases / metabolism*
  • Biotinylation
  • COS Cells
  • Cell Membrane / metabolism*
  • Cells, Cultured
  • Chlorocebus aethiops
  • Click Chemistry / methods*
  • Glycoproteins / metabolism*
  • Membrane Proteins / metabolism
  • Mice, Inbred C57BL
  • Neurons / metabolism*
  • Proteome / metabolism*
  • Reproducibility of Results
  • Substrate Specificity


  • Glycoproteins
  • Membrane Proteins
  • Proteome
  • Amyloid Precursor Protein Secretases
  • Aspartic Acid Endopeptidases
  • Bace1 protein, mouse