Sensitive and Specific Global Cell Surface N-Glycoproteomics Shows Profound Differences Between Glycosylation Sites and Subcellular Components
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Sensitive and Specific Global Cell Surface N-Glycoproteomics Shows Profound Differences Between Glycosylation Sites and Subcellular Components. / de Haan, Noortje; Song, Ming; Grant, Oliver C.; Ye, Zilu; Khoder Agha, Fawzi; Koed Møller Aasted, Mikkel; Woods, Robert J.; Vakhrushev, Sergey Y.; Wandall, Hans H.
I: Analytical Chemistry, Bind 95, Nr. 47, 2023, s. 17328-17336.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - Sensitive and Specific Global Cell Surface N-Glycoproteomics Shows Profound Differences Between Glycosylation Sites and Subcellular Components
AU - de Haan, Noortje
AU - Song, Ming
AU - Grant, Oliver C.
AU - Ye, Zilu
AU - Khoder Agha, Fawzi
AU - Koed Møller Aasted, Mikkel
AU - Woods, Robert J.
AU - Vakhrushev, Sergey Y.
AU - Wandall, Hans H.
N1 - Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
U2 - 10.1021/acs.analchem.3c03626
DO - 10.1021/acs.analchem.3c03626
M3 - Journal article
C2 - 37956981
AN - SCOPUS:85178379813
VL - 95
SP - 17328
EP - 17336
JO - Industrial And Engineering Chemistry Analytical Edition
JF - Industrial And Engineering Chemistry Analytical Edition
SN - 0003-2700
IS - 47
ER -
ID: 376567823