Sensitive and Specific Global Cell Surface N -Glycoproteomics Shows Profound Differences Between Glycosylation Sites and Subcellular Components

Analytical Chemistry, vol. 95, pp. 17328–17336

Abstract

High Resolution Image Download MS PowerPoint Slide Cell surface glycans are essential for establishing cell communication, adhesion, and migration. However, it remains challenging to obtain cell surface-specific information about glycoconjugate structures. Acquiring this information is essential for unraveling the functional role of glycans and for exploiting them as clinical targets. To specifically analyze the N -glycoprotein forms expressed at the cell surface, we developed a C18 liquid chromatography (LC)-mass spectrometry (MS)-based glycoproteomics method in combination with highly specific cell surface protein labeling and enrichment using a biotin label. The surface-specificity of the method was validated by MS-based proteomics of subcellular component marker proteins. Using the human keratinocytes N/TERT-1 as a model system, we identified and quantified the glycosylation of hundreds of cell surface N -glycosylation sites. This approach allowed us to study the glycoforms present at the functional relevant cell surface, omitting immaturely glycosylated proteins present in the secretory pathway. Interestingly, the different stages of N -glycan processing at individual sites displayed at the cell surface were found to correlate with their accessibility for ER-residing processing enzymes, as investigated through molecular dynamics simulations. Using the new approach, we compared N -glycosylation sites of proteins expressed on the cell surface to their counterparts in a total cell lysate, showing profound differences in glycosylation between the subcellular components and indicating the relevance of the method for future studies in understanding contextual glycan functions.

Authors 9

1. Noortje de Haan corresponding Aachen Center for Proteomics and Metabolomics

   University of Copenhagen · Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden 2333, ZA, The Netherlands

   Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen 2200, Denmark

2. Ming Song

   University of Copenhagen

   Affiliation as printed

   Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen 2200, Denmark

3. Oliver C. Grant

   University of Georgia

   Affiliation as printed

   Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States

4. Zilu Ye

   University of Copenhagen · Novo Nordisk Foundation

   Affiliation as printed

   Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen 2200, Denmark

5. Fawzi Khoder‐Agha

   University of Copenhagen

   Affiliation as printed

   Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen 2200, Denmark

6. Mikkel Koed Møller Aasted

   University of Copenhagen

   Affiliation as printed

   Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen 2200, Denmark

7. Robert J. Woods

   University of Georgia

   Affiliation as printed

   Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, United States

8. Sergey Y. Vakhrushev

   University of Copenhagen

   Affiliation as printed

   Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen 2200, Denmark

9. Hans Heugh Wandall corresponding

   University of Copenhagen

   Affiliation as printed

   Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen 2200, Denmark