İzzetoğlu S et al. (2014), Determination of sialic acids in immune system ...

ECB-ART-43110

Fish Shellfish Immunol 2014 Jan 01;361:181-6. doi: 10.1016/j.fsi.2013.10.029.

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Determination of sialic acids in immune system cells (coelomocytes) of sea urchin, Paracentrotus lividus, using capillary LC-ESI-MS/MS.

İzzetoğlu S , Şahar U , Şener E , Deveci R .

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Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac2, Neu5,7Ac2, Neu5,8Ac2, Neu5,7,9Ac3, Neu5Gc7,9Ac2, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).

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Genes referenced: LOC100887844

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References [+] :

Angata, Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. 2002, Pubmed

	Fig. 1. Schema of the reaction of 1,2-diamino-4,5-methylenediaoxy-benzene (DMB) derivatization of sialic acids.
	Fig. 2. LC-ESI-MS/MS mass spectrum and chromatogram of DMB-Sialic acid species (ten types) found in total coelomocyte population. Monitored of molecular ions [M + H]+ as labeled (♦) by software, and CID (collision-induced dissociation) fragments [M + H-H2O]+ at m/z; 424 for Neu5Gc, 408 for Neu5Ac, 466 for Neu5Gc7Ac, Neu5Gc8Ac and Neu5Gc9Ac, 450 for Neu5,7Ac2, Neu5,8Ac2 and Neu5,9Ac2, 492 for Neu5,7,9Ac3, 508 for Neu5Gc7,9Ac2.
	Fig. 3. LC-ESI-MS/MS mass spectrum and chromatogram of DMB-Standard Sialic acid species (KDN, Neu5Gc and Neu5Ac). Monitored of molecular ions [M + H]+ as labeled (♦) by software, and CID (collision-induced dissociation) fragments [M + H-H2O]+ at m/z.
	Fig. 4. Distribution of sialic acids amounts measured by LC-ESI-MS/MS. Sialic acid contents of total coelomocyte population were demonstrated by average peak area of MRM chromatograms of their types. Data are presented as mean ± standard deviation (S.D.) of peak area from three replicated experiments.