HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M800272-MCP200v1 8/2/287    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via Google Scholar Google Scholar Articles by McDonald, C. A. Articles by Macher, B. A. Search for Related Content PubMed PubMed Citation Articles by McDonald, C. A. Articles by Macher, B. A. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:287-301, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

Combining Results from Lectin Affinity Chromatography and Glycocapture Approaches Substantially Improves the Coverage of the Glycoproteome^*,S

Claudia A. McDonald, Jane Y. Yang, Vinita Marathe, Ten-Yang Yen and Bruce A. Macher

From the Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometry-based analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycoproteins. The plasma membrane localization of a subset of proteins identified was validated, and the dynamics of their expression in HeLa cells was evaluated during the cell cycle. Results obtained from the cell cycle studies demonstrate that plasma membrane protein expression can change up to 4-fold as cells transit the cell cycle and demonstrate the need to consider such changes when carrying out quantitative proteomics comparison of cell lines.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?