DOES MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY ALLOW ANALYSIS OF CARBOHYDRATE HETEROGENEITY IN GLYCOPROTEINS - A STUDY OF NATURAL HUMAN INTERFERON-GAMMA

Interferon-gamma (IFN-gamma) is a dimeric, secretory glycoprotein prod uced by T-lymphocytes. The glycan microheterogeneity of natural human IFN-gamma was characterized by matrix-assisted laser desorption/ioniza tion mass spectrometry (MALDI/MS) combined with glycosidase digestion. The glycan structures at the two potential glycosylation sites, aspar agine 25 and 97, differ in composition and heterogeneity. The glycan a t Asn 25 consists of a mixture of hybrid structures and fucosylated co mplex bi-, tri- and tetra-antennary structures, whereas the glycan at Asn 97 is more heterogeneous and consists of a mixture of high mannose structures, hybrid structures and unfucosylated complex bi- and tri-a ntennary structures. The contribution to the observed glycan heterogen eity by prompt and metastable fragmentation was evaluated by treatment s with different exoglycosidases and by comparison of linear, reflecte d and delayed extraction MALDI/TOF mass spectra, Heterogeneity observe d with the matrices alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydroxyb enzoic acid and 2,4,6-trihydroxyacetophenone was compared. Most of the heterogeneity can be attributed to native structure diversity and onl y to a minor extent to mass spectrometric fragmentation such as fragme ntational loss of sialic acid residues.