title: Rapid analysis of cell surface N-glycosylation from living cells using mass spectrometry.
abstract: Cell surfaces are covered with a dense carbohydrate layer referred to as the glycocalyx. Because different cell types express different glycan signatures, it is of paramount importance to have robust methods to analyze the glycome of living cells. To achieve this, a common procedure involves cell lysis and extraction of membrane (glyco)proteins and yields a major proportion of high-mannose N-glycans that most likely stem from intracellular proteins derived from the ER. Using HEK 293 cells as a model system, we developed a reproducible, sensitive, and fast method to profile surface N-glycosylation from living cells. We directly released glycopeptides from cell surfaces through tryptic digestion of freshly harvested and vital cells, thereby improving the detection and quantification of complex-type N-glycans by increasing their relative amount from 14 to 85%. It was also possible to detect 25 additional structures in HEK 293, 48 in AGE1.HN, 42 in CHO-K1, and 51 in Hep G2 cells. The additional signals provided deeper insight into cell-type-specific N-glycan features such as antennarity, fucosylation, and sialylation. Thus, this protocol, which can potentially be applied to any cells, will be useful in the fields of glycobiotechnology and biomarker discovery.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin , Augustenburger Platz 1, 13353 Berlin, Germany.
authors.forename: Houda
authors.initials: H
authors.lastname: Hamouda
authors.forename: Matthias
authors.initials: M
authors.lastname: Kaup
authors.forename: Mujib
authors.initials: M
authors.lastname: Ullah
authors.forename: Markus
authors.initials: M
authors.lastname: Berger
authors.forename: Volker
authors.initials: V
authors.lastname: Sandig
authors.forename: Rudolf
authors.initials: R
authors.lastname: Tauber
authors.forename: Véronique
authors.initials: V
authors.lastname: Blanchard
mesh_terms: D000818:Animals; D016466:CHO Cells; D002457:Cell Extracts / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006020:Glycopeptides / Q000032:analysis* / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D011134:Polysaccharides / Q000032:analysis / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D020543:Proteome; D006026:Glycoside Hydrolases; D014357:Trypsin
keywords: Cell surface; MALDI-TOF-MS; N-glycosylation; glycocalyx