New sialic acids from biological sources identified by a comprehensive and sensitive approach: Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) of SIA quinoxalinones

Sialic acids are a family of 9-carbon carboxylated sugars, where different substitutions of the backbone define over 30 members. Biological roles of these substitutions have been missed until recently because of their low abundance and lability to conventional isolation/purification methods. This new approach characterizes sialic acids using electrospray ionization-mass spectrometry (ESI-MS) to monitor the HPLC separation of their DMB (1,2-diamino-4,5-methylenedioxy-benzene) derivatives (quinoxalinones). A combination of retention times and spectra characteristics allows definition of the type and position of the various substituents. This approach requires no previous purification, involving a simple derivatization reaction followed by direct injection on the microbore HPLC column. A complete spectrum, including molecular ions and CAD fragments of a sialic acid quinoxalinone, is obtained by injecting 10-20 pmol of the compound. Individual quinoxalinones can be purified by regular RP-HPLC and analyzed by direct-injection ESI-MS or LSIMS. Using this approach, we identified 28 different sialic acids, including the following new species: Neu5Gc9Lt (BSM), anhydro derivatives of Neu5Ac other than the 4,8-anhydro (horse serum hydrolyzates), KDN5(7)Ac and KDN5(7),9Ac(2) (amphibian Pleurodeles waltl), four isomers of Neu5Gc8MexAc and three anhydro derivatives of Neu5Gc8Me (glycolipids of the starfish Pisaster brevispinus), and Neu5Ac8S (in addition to Neu5Gc8S, in the glycolipids of the sea urchin Lovenia cordiformis). Results show the usefulness of LC-ESIMS to study sialic acid diversity, and identification of small amounts of unexpected sialic acids or new members of their family.