Determination of the type and quantity of sialic acid in the egg jelly coat of the sea urchin Paracentrotus lividus using capillary LC-ESI-MS/MS

SUMMARY Sialic acid is a terminal sugar of carbohydrate chains that participates in numerous biological events. Recent studies have explored the mechanism of carbohydrate‐mediated fertilisation to understand the biochemistry of fertilisation, although the type and quantity of sialic acid and the role of sialic acid during fertilisation remain unknown. Echinoderm fertilisation in particular has been studied extensively, yet our understanding of the mechanisms of carbohydrate‐mediated fertilisation and the role of sialic acid remains incomplete. In this study, we characterised the sialic acid types in the egg jelly coat of the sea urchin, Paracentrotus lividus, using the sensitive analytical system capillary liquid chromatography electro‐spray ionisation tandem mass spectrometry (capLC‐ESI‐MS/MS). First, we isolated the egg jelly coat and released its sialic acid using acid treatment. These sialic acids were derivatised with 1,2‐diamino‐4,5‐methylenediaoxy‐benzene dihydrochloride (DMB) and injected into the capLC‐ESI‐MS/MS system. When compared with standards, we identified twelve different types of sialic acid according to their retention times and collision‐induced dissociation fragments. The mass spectral data revealed that Neu5Gc, Neu5Ac, Neu5GcS, and Neu5Gc9Ac were the predominant types of sialic acid in the sea urchin jelly coat, with Neu5Gc being the most abundant. Other types of sialic acid detected included Neu5AcS, Neu5Gc7,9Ac2, Neu5,9Ac2, Neu5Gc8Ac, Neu5Gc7Ac, Neu5,7Ac2, Neu5Gc8,9Ac2, and Neu5,8Ac2. The types and quantities of sialic acid that we detected in the egg jelly coat will aid in the discovery of new sialic acid‐specific receptors on the sperm membrane. Mol. Reprod. Dev. 82: 115–122, 2015. © 2014 Wiley Periodicals, Inc.