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Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection.
Electrophoresis. 2008 Nov; 29(22):4538-48.E

Abstract

This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.

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Authors+Show Affiliations

Hitchcock AM
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA.
Bowman MJ
No affiliation info available
Staples GO
No affiliation info available
Zaia J
No affiliation info available

MeSH

AminoacridinesAnimalsCartilageCattleCelluloseChondroitin SulfatesDermatan SulfateDisaccharidesElectrophoresis, CapillaryFluorescenceGlycosaminoglycansHeparinHeparitin SulfateHumansLyasesReproducibility of ResultsSensitivity and Specificity

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

19035406

Citation

Hitchcock, Alicia M., et al. "Improved Workup for Glycosaminoglycan Disaccharide Analysis Using CE With LIF Detection." Electrophoresis, vol. 29, no. 22, 2008, pp. 4538-48.
Hitchcock AM, Bowman MJ, Staples GO, et al. Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection. Electrophoresis. 2008;29(22):4538-48.
Hitchcock, A. M., Bowman, M. J., Staples, G. O., & Zaia, J. (2008). Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection. Electrophoresis, 29(22), 4538-48. https://doi.org/10.1002/elps.200800335
Hitchcock AM, et al. Improved Workup for Glycosaminoglycan Disaccharide Analysis Using CE With LIF Detection. Electrophoresis. 2008;29(22):4538-48. PubMed PMID: 19035406.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection. AU - Hitchcock,Alicia M, AU - Bowman,Michael J, AU - Staples,Gregory O, AU - Zaia,Joseph, PY - 2008/11/28/pubmed PY - 2009/4/21/medline PY - 2008/11/28/entrez SP - 4538 EP - 48 JF - Electrophoresis JO - Electrophoresis VL - 29 IS - 22 N2 - This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue. SN - 1522-2683 UR - https://www.unboundmedicine.com/medline/citation/19035406/Improved_workup_for_glycosaminoglycan_disaccharide_analysis_using_CE_with_LIF_detection_ L2 - https://doi.org/10.1002/elps.200800335 DB - PRIME DP - Unbound Medicine ER -