Comprehensive profiling of N-linked glycosylation sites in HeLa cells using hydrazide enrichment

Helle Malerod, Robert L J Graham, Michael J Sweredoski, Sonja Hess

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)248-59
Number of pages12
JournalJournal of Proteome Research
Volume12
Issue number1
DOIs
Publication statusPublished - 04 Jan 2013

Keywords

  • Asparagine/metabolism
  • Biomarkers, Tumor/genetics
  • Chromatography, Liquid
  • Female
  • Glycoproteins/genetics
  • Glycosylation
  • HeLa Cells/metabolism
  • Humans
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism
  • Polysaccharides/metabolism
  • Tandem Mass Spectrometry
  • Uterine Cervical Neoplasms/genetics

Fingerprint Dive into the research topics of 'Comprehensive profiling of N-linked glycosylation sites in HeLa cells using hydrazide enrichment'. Together they form a unique fingerprint.

  • Cite this