The haemoglobins (Hbs) of aquatic and terrestrial annelids have a characteristic hexagonal bilayer electron microscopic appearance, a high mass (approx 3.5MDa) and consists of about l44 functional globin chains (16-1 8kDa) accounting for about 75% of the total mass and some 36-40 linker CHAINS (24-32 kDa) necessary for the formation of the native structure. Some of the different globin chains are monomeric but others form disulphide-bonded trimers (earthworm Hb) or dimers (leech Hb). In addition, some globin and linker chains are glycosylated depending on the species. The mixture of proteins and glycoproteins produced from earth worm Hb contains about 15 components with molecular weights between 16 and 53 kDa.

We have employed electrospray ionisation mass spectrometry (ESI-MS) with maximum entropy processing of the original data, to analyse the complex mixtures of proteins produced directly from various annelid Hbs. In this way, rapid surveys of the masses (within 0.0l%) of all those proteins which are non-covalently bound to one another in the native Hb were produced without prior separation. These survey spectra clearly resolved glycosylated components up to 52 kDa and indicated which chains were glycosylated by revealing two or more peaks related by sugar residue mass differences, produced as a result of heterogeneity in the glycan. Reduction of the dimers and trimers with or without carboxymethylation, allowed the masses of their constituent chains to be established unambiguously.

Furthermore, the application of ESI-MS-MS to certain glycoproteins enabled their glycan composition to be determined. Data from several annelid Hbs will be presented.