Improving size-exclusion chromatography separation for glycogen

Sullivan, Mitchell A., Powell, Prudence O., Witt, Torsten, Vilaplana, Francisco, Roura, Eugeni and Gilbert, Robert G. (2014) Improving size-exclusion chromatography separation for glycogen. Journal of Chromatography A, 1332 21-29. doi:10.1016/j.chroma.2014.01.053

Author Sullivan, Mitchell A.
Powell, Prudence O.
Witt, Torsten
Vilaplana, Francisco
Roura, Eugeni
Gilbert, Robert G.
Title Improving size-exclusion chromatography separation for glycogen
Journal name Journal of Chromatography A   Check publisher's open access policy
ISSN 0021-9673
Publication date 2014-03-07
Year available 2014
Sub-type Article (original research)
DOI 10.1016/j.chroma.2014.01.053
Open Access Status Not yet assessed
Volume 1332
Start page 21
End page 29
Total pages 9
Place of publication Amsterdam, Netherlands
Publisher Elsevier
Language eng
Subject 1602 Analytical Chemistry
1605 Organic Chemistry
1303 Biochemistry
Abstract Exosomes are a subset of extracellular vesicles (EVs) that have important roles in intercellular communication. They contain and carry bioactive molecules within their membranes which are delivered to target cells. Reproducible isolation and enrichment of these exosomes will aid in evaluation of cellular communication. We present an approach that involved the pre-processing of plasma, combined with ultracentrifugation (UC) and size exclusion chromatography (SEC) to isolate EVs and subsequently enrich exosomes. Four variations of this approach (denoted methods I to IV) were compared. Coupling an ultracentrifugation method with size exclusion chromatography (Method II) provided the best yield by nanoparticle tracking analyses (NTA), the presence of the exosomal markers CD63, Flotillin-1 and TSG-101 (immunoblotting) and showed exosome morphology using transmission electron microscopy (TEM). This method provides an efficient way to enrich the exosomes from blood (plasma), which could be potentially employed for clinical diagnostic assessment and therapeutic intervention.
Formatted abstract
Glycogen is a hyperbranched glucose polymer comprised of glycogen β particles, which can also form much larger composite α particles. The recent discovery using size-exclusion chromatography (SEC) that fewer, smaller, α particles are found in diabetic-mouse liver compared to healthy mice highlights the need to achieve greater accuracy in the size separation methods used to analyze α and β particles. While past studies have used dimethyl sulfoxide as the SEC eluent to analyze the molecular size and structure of native glycogen, an aqueous eluent has not been rigorously tested and compared with dimethyl sulfoxide. The conditions for SEC of pig-liver glycogen, phytoglycogen and oyster glycogen were optimized by comparing two different eluents, aqueous 50mM NH4NO3/0.02% NaN3 and dimethyl sulfoxide/0.5% LiBr, run through different column materials and pore sizes at various flow rates. The aqueous system gave distinct size separation of α- and β-particle peaks, allowing for a more detailed and quantitative analysis and comparison between liver glycogen samples. This greater resolution has also revealed key differences between the structure of liver glycogen and phytoglycogen.
Keyword Glycogen
Improved resolution
Size-exclusion chromatography (SEC)
Structural characterization
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Queensland Alliance for Agriculture and Food Innovation
Official 2015 Collection
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Citation counts: TR Web of Science Citation Count  Cited 16 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 14 times in Scopus Article | Citations
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Created: Tue, 11 Mar 2014, 10:20:32 EST by System User on behalf of Centre for Nutrition and Food Sciences