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Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring.
Anal Biochem. 2017 08 01; 530:17-30.AB

Abstract

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 μg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 μg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

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Authors+Show Affiliations

Antia IU
Glycan Research Group, Department of Natural Sciences, Faculty of Science and Technology, Middlesex University, The Burroughs, London NW4 4BT, UK. Electronic address: i.antia@mdx.ac.uk.
Yagnik DR
Glycan Research Group, Department of Natural Sciences, Faculty of Science and Technology, Middlesex University, The Burroughs, London NW4 4BT, UK. Electronic address: d.yagnik@mdx.ac.uk.
Pantoja Munoz L
Glycan Research Group, Department of Natural Sciences, Faculty of Science and Technology, Middlesex University, The Burroughs, London NW4 4BT, UK. Electronic address: L.PantojaMunoz@mdx.ac.uk.
Shah AJ
Glycan Research Group, Department of Natural Sciences, Faculty of Science and Technology, Middlesex University, The Burroughs, London NW4 4BT, UK. Electronic address: a.j.shah@mdx.ac.uk.
Hills FA
Glycan Research Group, Department of Natural Sciences, Faculty of Science and Technology, Middlesex University, The Burroughs, London NW4 4BT, UK. Electronic address: f.hills@mdx.ac.uk.

MeSH

AminoacridinesChromatography, High Pressure LiquidDisaccharidesHeparinHeparin LyaseHeparitin SulfateHumansMass SpectrometryNeoplasmsTumor Cells, Cultured

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28465034

Citation

Antia, Imeobong U., et al. "Heparan Sulfate Disaccharide Measurement From Biological Samples Using Pre-column Derivatization, UPLC-MS and Single Ion Monitoring." Analytical Biochemistry, vol. 530, 2017, pp. 17-30.
Antia IU, Yagnik DR, Pantoja Munoz L, et al. Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring. Anal Biochem. 2017;530:17-30.
Antia, I. U., Yagnik, D. R., Pantoja Munoz, L., Shah, A. J., & Hills, F. A. (2017). Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring. Analytical Biochemistry, 530, 17-30. https://doi.org/10.1016/j.ab.2017.04.019
Antia IU, et al. Heparan Sulfate Disaccharide Measurement From Biological Samples Using Pre-column Derivatization, UPLC-MS and Single Ion Monitoring. Anal Biochem. 2017 08 1;530:17-30. PubMed PMID: 28465034.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring. AU - Antia,Imeobong U, AU - Yagnik,Darshna R, AU - Pantoja Munoz,Leonardo, AU - Shah,Ajit J, AU - Hills,Frank A, Y1 - 2017/04/30/ PY - 2017/01/24/received PY - 2017/03/06/revised PY - 2017/04/27/accepted PY - 2017/5/4/pubmed PY - 2017/8/18/medline PY - 2017/5/4/entrez KW - 2- aminoacridone KW - Cancer cell lines KW - Glycosaminoglycan KW - Heparan sulfate KW - Proteoglycan KW - RP-UPLC-MS SP - 17 EP - 30 JF - Analytical biochemistry JO - Anal Biochem VL - 530 N2 - Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 μg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 μg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences. SN - 1096-0309 UR - https://www.unboundmedicine.com/medline/citation/28465034/Heparan_sulfate_disaccharide_measurement_from_biological_samples_using_pre_column_derivatization_UPLC_MS_and_single_ion_monitoring_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(17)30187-2 DB - PRIME DP - Unbound Medicine ER -
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