Glycosyltransferases appear to be highly specific and the structural specificity is primarily retained by the oligosaccharide. Tbus, from the limited studies described, it appears that a residue's position and linkage follows a specific pattern and glycoprotein heterogeneity represents a lack of pattern completion. Fundamental to understanding glycan functional significance is a detailed site-specific structural analysis. To approach this first problem, we have undertaken glycan molecular weight profiles on the protein constructs LFA3 and IgG. LFA3TIP wasconstructed with the first extracellular domain of lymphocyte function-associated molecule 3 (LFA3) fused to the hinge, CH2, and CH3 regions of a human IgGl. Analyses were acquired by endoglycosidase release of N-linked glycans, methylation, and extraction using electrospray ionization mass spectrometry and collision-induced decomposition (CID). In order to determine neuraminyl linkages (e.g., 2-+3, vs. 2-+6), lactosyl extensions, and triantennary branched positions, oxidation, reduction, and methylation (ORM) were carried out on the released glycans prior to ES-MS and CID. Preliminary assessment of enzymatic release strategies, methylation and extraction were evaluated by ESI MS on samples LFA3-A, LFA3-Al, LFA3113, and LFA3 pg of sample each of LFA3-A (TS95RO275) and LFA3-B (TS95RO275). These studies have shown that the terminating sugars are not randomly distributed over the various antennae, but occur preferentially attached to specific N-acetyllactosamine residues. This ESI MS mass profile of all structures can be most informative and serves as a first level of structural inquiry, akin to carbohydrate composition analysis.
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