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Comprehensive Profiling of N‑Linked Glycosylation Sites in HeLa Cells Using Hydrazide Enrichment

Malerod, Helle and Graham, Robert L. J. and Sweredoski, Michael J. and Hess, Sonja (2013) Comprehensive Profiling of N‑Linked Glycosylation Sites in HeLa Cells Using Hydrazide Enrichment. Journal of Proteome Research, 12 (1). pp. 248-259. ISSN 1535-3893. doi:10.1021/pr300859k. https://resolver.caltech.edu/CaltechAUTHORS:20130301-124120701

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/pr300859kDOIArticle
http://pubs.acs.org/doi/abs/10.1021/pr300859kPublisherArticle
ORCID:
AuthorORCID
Sweredoski, Michael J.0000-0003-0878-3831
Hess, Sonja0000-0002-5904-9816
Additional Information:© 2012 American Chemical Society. Published In Issue January 04, 2013; Article ASAP December 04, 2012; Just Accepted Manuscript November 23, 2012; Received: July 18, 2012. The Norwegian Research council (Project 197431/F20) (H.M.), the Faculty of Mathematics and Natural Sciences at the University of Oslo (H.M.), the Betty and Gordon Moore Foundation (R.L.J.G., M.S., S.H.), and the Beckman Institute (M.S., S.H.) are acknowledged for their financial support. We thank Ruth Huettenhain (ETHZ) for critical reading of the manuscript. The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
Norwegian Research Council197431/F20
University of OsloUNSPECIFIED
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Subject Keywords:N-linked glycoproteins; hydrazide capture; HeLa cells; cancer; biomarkers
Issue or Number:1
DOI:10.1021/pr300859k
Record Number:CaltechAUTHORS:20130301-124120701
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20130301-124120701
Official Citation:Comprehensive Profiling of N-Linked Glycosylation Sites in HeLa Cells Using Hydrazide Enrichment Helle Malerod, Robert L. J. Graham, Michael J. Sweredoski, and Sonja Hess Journal of Proteome Research 2013 12 (1), 248-259
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:37250
Collection:CaltechAUTHORS
Deposited By: Aucoeur Ngo
Deposited On:02 Mar 2013 00:02
Last Modified:09 Nov 2021 23:27

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