Immunoglobulin A carries sulfated and -acetylated -glycans primarily at the tailpiece site - strategies for site-specific -glycan identification.

Sulfated glycans from human immunoglobulin A (IgA) were recently discovered via glycomic approaches. However, their site-specific description is still pending. Certain glycan structures at specific glycosylation sites in IgA are crucial for microbial neutralization and effector functions. For instance, sialylated glycans on the C-terminal tailpiece mediate anti-viral activity by interfering with sialic-acid-binding viruses. Sulfated glycan epitopes can be ligands for viral proteins and thus play a role in the immune response. In this study, we performed a site-specific screening for sulfated and other rare glycans in two commercially available human serum IgA samples employing an in-depth glycoproteomic approach, previously developed by us. We found evidence of complex-type and hybrid-type glycans containing sulfated acetylhexosamine (sulfated HexNAc) attached to the glycosylation sites in the tailpiece and the C2 domain of both IgA subclasses. Also, complex-type glycan compositions bearing acetylated sialic acid were identified primarily at the tailpiece site. Surprisingly, glycans bearing glucuronic acid were identified in the commercial IgA samples, but from peptides of "contaminant" glycoproteins. A detailed comparison of the glycosylation profiles of human serum IgA samples from two suppliers showed such glycans with sulfated HexNAc consistently in higher abundance in the tailpiece region. These findings have not been described before for a site-specific glycopeptide analysis. Overall, our work provides strategies for performing a dedicated site-specific search for sulfated and acetylated glycans that can be easily transferred, e.g., to human IgA derived from mucosal tissues, milk, or saliva. We expect that a wider and deeper micro-heterogeneity description of clinically relevant glycoproteins, such as immunoglobulins, can expand the screening for biomarkers or treatment options.