Oligosaccharide mapping based on enzyme cleavage provides a useful molecular fingerprint of the heparin structure revealing detailed structural information regarding its sequence and the content of part of the ATIII-binding region. This approach is performed by strong-anion exchange (SAX)-HPLC separation which is incompatible with MS requiring purification of oligosaccharides for their conclusive identification. We report a novel oligosaccharide mapping strategy based on the HILIC separation of the main heparin disaccharides/oligosaccharides released by heparinase I, fluorotagged with 2-aminoacridone and on-line detected by a fluorescence detector and characterized by ESI-MS. The application of a polar solvent having a high pH with acetonitrile avoided desulfation enabling a simple and accurate structural oligosaccharide assignment. Oligosaccharide mapping, or merely complete disaccharide composition, may be performed on nanogram-scale by the fluorescence detector vs micrograms useful for classical SAX-HPLC. Additionally, only widely commercially available heparin lyase I is necessary, without the use of expensive heparinases II and III. Contrary to SAX-HPLC, this novel HILIC approach is able to separate and identify the saturated trisulfated disaccharide belonging to the non-reducing end of heparin chains. Finally, the content of 3-O-sulfo groups of the ATIII-binding region is determined.

Oligosaccharide mapping of heparinase I-treated heparins by hydrophilic interaction liquid chromatography separation and online fluorescence detection and electrospray ionization-mass spectrometry characterization / Galeotti, Fabio; Volpi, Nicola. - In: JOURNAL OF CHROMATOGRAPHY A. - ISSN 0021-9673. - STAMPA. - 1445:(2016), pp. 68-79. [10.1016/j.chroma.2016.03.078]

Oligosaccharide mapping of heparinase I-treated heparins by hydrophilic interaction liquid chromatography separation and online fluorescence detection and electrospray ionization-mass spectrometry characterization.

GALEOTTI, FABIO;VOLPI, Nicola
2016

Abstract

Oligosaccharide mapping based on enzyme cleavage provides a useful molecular fingerprint of the heparin structure revealing detailed structural information regarding its sequence and the content of part of the ATIII-binding region. This approach is performed by strong-anion exchange (SAX)-HPLC separation which is incompatible with MS requiring purification of oligosaccharides for their conclusive identification. We report a novel oligosaccharide mapping strategy based on the HILIC separation of the main heparin disaccharides/oligosaccharides released by heparinase I, fluorotagged with 2-aminoacridone and on-line detected by a fluorescence detector and characterized by ESI-MS. The application of a polar solvent having a high pH with acetonitrile avoided desulfation enabling a simple and accurate structural oligosaccharide assignment. Oligosaccharide mapping, or merely complete disaccharide composition, may be performed on nanogram-scale by the fluorescence detector vs micrograms useful for classical SAX-HPLC. Additionally, only widely commercially available heparin lyase I is necessary, without the use of expensive heparinases II and III. Contrary to SAX-HPLC, this novel HILIC approach is able to separate and identify the saturated trisulfated disaccharide belonging to the non-reducing end of heparin chains. Finally, the content of 3-O-sulfo groups of the ATIII-binding region is determined.
2016
1-apr-2016
1445
68
79
Oligosaccharide mapping of heparinase I-treated heparins by hydrophilic interaction liquid chromatography separation and online fluorescence detection and electrospray ionization-mass spectrometry characterization / Galeotti, Fabio; Volpi, Nicola. - In: JOURNAL OF CHROMATOGRAPHY A. - ISSN 0021-9673. - STAMPA. - 1445:(2016), pp. 68-79. [10.1016/j.chroma.2016.03.078]
Galeotti, Fabio; Volpi, Nicola
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1110793
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