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 diffe rent substitutions of the backbone define over 30 members. Biological roles of these substitutions have been missed until recently because o f their low abundance and lability to conventional isolation/purificat ion methods. This new approach characterizes sialic acids using electr ospray ionization-mass spectrometry (ESI-MS) to monitor the HPLC separ ation of their DMB (1,2-diamino-4,5-methylenedioxy-benzene) derivative s (quinoxalinones). A combination of retention times and spectra chara cteristics allows definition of the type and position of the various s ubstituents. This approach requires no previous purification, involvin g 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 injec ting 10-20 pmol of the compound. Individual quinoxalinones can be puri fied by regular RP-HPLC and analyzed by direct-injection ESI-MS or LSI MS. Using this approach, we identified 28 different sialic acids, incl uding 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 Ne u5Gc8S, in the glycolipids of the sea urchin Lovenia cordiformis). Res ults show the usefulness of LC-ESIMS to study sialic acid diversity, a nd identification of small amounts of unexpected sialic acids or new m embers of their family.