title: Comprehensive profiling of N-linked glycosylation sites in HeLa cells using hydrazide enrichment.
abstract: The adenocarcinoma cell line HeLa serves as a model system for cancer research in general and cervical cancer in particular. In this study, hydrazide enrichment in combination with state-of-the art nanoLC-MS/MS analysis was used to profile N-linked glycosites in HeLa cells. N-Linked glycoproteins were selectively enriched in HeLa cells by the hydrazide capture method, which isolates all glycoproteins independent of their glycans. Nonglycosylated proteins were removed by extensive washing. N-Linked glycoproteins were identified with the specific NXT/S motif and deamidated asparagine (N). Deglycosylation was carried out in both H(2)(16)O and H(2)(18)O to confirm the deamidation. NanoLC-MS/MS analysis indicated that the method selectively enriched at least 100 fold N-linked glycosites in HeLa cells. When both the membrane and cytosolic fractions were used, a total of 268 unique N-glycosylation sites were identified corresponding to 106 glycoproteins. Bioinformatic analysis revealed that most of the glycoproteins identified are known to have an impact on cancer and have been proposed as biomarkers.
authors.affiliation: Department of Chemistry, University of Oslo, PO Box 1033 Blindern, 0315 Oslo, Norway.
authors.forename: Helle
authors.initials: H
authors.lastname: Malerod
authors.forename: Robert L J
authors.initials: RL
authors.lastname: Graham
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
mesh_terms: D001216:Asparagine / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid; D005260:Female; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells / Q000378:metabolism / Q000473:pathology; D006801:Humans; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D006023:Glycoproteins; D011134:Polysaccharides; D001216:Asparagine; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase