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 produced by T-lymphocytes. The glycan micro-heterogeneity of natural human IFN-gamma was characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) combined with glycosidase digestion. The glycan structures at the two potential glycosylation sites, asparagine 25 and 97, differ in composition and heterogeneity. The glycan at Asn 25 consists of a mixture of hybrid structures and fucosylated complex 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-antennary structures. The contribution to the observed glycan heterogeneity by prompt and metastable fragmentation was evaluated by treatments with different exoglycosidases and by comparison of linear, reflected and delayed extraction MALDI/TOF mass spectra. Heterogeneity observed with the matrices alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid and 2,4,6-trihydroxyacetophenone was compared. Most of the heterogeneity can be attributed to native structure diversity and only to a minor extent to mass spectrometric fragmentation such as fragmentational loss of sialic acid residues.