IgA is the most abundantly produced antibody and plays an important role in the 
mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. 
The major structural difference between these two subclasses is the presence of 
nine potential sites of O-glycosylation in the hinge region between the first 
and second constant region domains of the heavy chain. Thr(225), Thr(228), 
Ser(230), Ser(232) and Thr(236) have been identified as the predominant sites of 
O-glycan attachment. The range and distribution of O-glycan chains at each site 
within the context of adjacent sites in this clustered region create a complex 
heterogeneity of surface epitopes that is incompletely defined. We previously 
described the analysis of IgA1 O-glycan heterogeneity by use of high resolution 
LC-MS and electron capture dissociation tandem MS to unambiguously localize all 
amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the 
identification and elucidation of IgA1 O-glycopeptide structural isomers that 
occur based on amino acid position of the attached glycans (positional isomers) 
and the structure of the O-glycan chains at individual sites (glycan isomers). 
These isomers are present in a model IgA1 (Mce1) myeloma protein and occur 
naturally in normal human serum IgA1. Variable O-glycan chains attached to 
Ser(230), Thr(233) or Thr(236) produce the predominant positional isomers, 
including O-glycans composed of a single GalNAc residue. These findings 
represent the first definitive identification of structural isomeric IgA1 
O-glycoforms, define the single-site heterogeneity for all O-glycan sites in a 
single sample, and have implications for defining epitopes based on clustered 
O-glycan variability.