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Sequence determination of a non-sulfated glycosaminoglycan-like polysaccharide from melanin-free ink of the squid Ommastrephes bartrami by negative-ion electrospray tandem mass spectrometry and NMR spectroscopy

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Abstract

A non-sulfated polysaccharide was isolated from the ink sac of squid Ommastrephes bartrami after removal of the melanin granules. The carbohydrate sequence of this polysaccharide was assigned by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation of the oligosaccharide fractions produced by partial acid hydrolysis of the polysaccharide. The structural determination was completed by NMR for assignment of anomeric configuration and confirmation of linkage information and it was unambiguously identified as a glycosaminoglycan-like polysaccharide containing a glucuronic acid–fucose (GlcA-Fuc) disaccharide repeat in the main chain and a N-acetylgalactosamine (GalNAc) branch at Fuc position 3: –[3GlcAβ1–4(GalNAcα1–3)Fucα1]n–. Partial hydrolysis of the polysaccharide to obtain several oligosaccharide fractions with different numbers of the repeating unit assisted the assignment. In the negative-ion tandem mass spectrometric analysis, the unique 0,2A type fragmentation was important to establish the presence of a 4-linked fucose in the main polysaccharide chain and a GalNAc branch at the Fuc position–3 of the disaccharide repeat.

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Abbreviations

ES-MS:

electrospray mass spectrometry

CID:

collision-induced dissociation

COSY:

correlation spectroscopy

HMBC:

heteronuclear multiple bond correlation

HMQC:

heteronuclear single quantum coherence

TFA:

trifluoroacetic acid

PMP:

1-phenyl-3-methyl-5-pyrazolone

GAG:

glycosaminoglycan

SIP:

squid ink polysaccharide

SIO:

squid ink oligosaccharide

Fuc:

Fucose

Ara:

arabinose

Glc:

glucose

Man:

mannose

Gal:

galactose

GalN:

galactosamine

GlcN:

glucosamine

GlcA:

glucuronic acid

GalA:

galacturonic acid

GalNAc:

N-acetylgalactosamine

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Acknowledgements

This work was supported in part by the program ‘New Century Excellent Talents in University’ (NCET-04-0642), National Natural Science Foundation of China (2006AA09Z444), OUC Lvka Program (1405-814147), an international collaboration grant from the Ministry of Science and Technology (2007DFA30980) and a U.K. Medical Research Council grant (G0600512). We thank Shumei Ren for ES-MS experiments. NMR was carried out at the College of Medicine and Pharmacy, Ocean University China.

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Authors and Affiliations

  1. College of Food Science and Technology, Ocean University of China, 5 Yu Shan Road, Qingdao, Shandong, 266003, China

    Shiguo Chen, Jie Xu, Changhu Xue, Ping Dong & Wenjing Sheng

  2. College of Medicine and Pharmacy, Ocean University of China, Qingdao, China

    Guangli Yu & Wengang Chai

  3. Glycosciences Laboratory, Imperial College Faculty of Medicine, Northwick Park and St. Mark’s Campus, Harrow, Middlesex, HA1 3UJ, UK

    Wengang Chai

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  1. Shiguo Chen
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Correspondence to Changhu Xue or Wengang Chai.

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Chen, S., Xu, J., Xue, C. et al. Sequence determination of a non-sulfated glycosaminoglycan-like polysaccharide from melanin-free ink of the squid Ommastrephes bartrami by negative-ion electrospray tandem mass spectrometry and NMR spectroscopy. Glycoconj J 25, 481–492 (2008). https://doi.org/10.1007/s10719-007-9096-2

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