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J Vis Exp. 2014 Nov 20;(93):e51840. doi: 10.3791/51840.

Improved in-gel reductive β-elimination for comprehensive O-linked and sulfo-glycomics by mass spectrometry.

Author information

1
Complex Carbohydrate Research Center, University of Georgia; Department of Biochemistry and Molecular Biology, University of Georgia.
2
Complex Carbohydrate Research Center, University of Georgia.
3
Complex Carbohydrate Research Center, University of Georgia; Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University.
4
Complex Carbohydrate Research Center, University of Georgia; kaoki@ccrc.uga.edu.

Abstract

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive β-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

PMID:
25489664
PMCID:
PMC4354198
DOI:
10.3791/51840
[Indexed for MEDLINE]
Free PMC Article

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