Insight into distribution and composition of nonhuman N-Glycans in mammalian organs via MALDI-TOF and MALDI-MSI.

The major hurdle of xenotransplantation is the immune response triggered by human natural antibodies interacting with carbohydrate antigens on the transplanted animal organ. Specifically, terminal glycoprotein motifs such as galactose-α1,3-galactose (α-Gal) and N-glycolylneuraminic acid (Neu5Gc) are significant obstacles. Little is known about the abundance and compositions of asparagine-linked complex carbohydrates (N-glycans) carrying these motifs in mammalian organs. By studying heart, kidney, and liver tissues from pig, cattle, and sheep, we aimed to gain insights into the abundance and spatial distribution of α-Gal- or Neu5Gc-containing N-glycans. N-glycomes were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), MALDI-mass spectrometry imaging (MSI), and capillary electrophoresis-electrospray ionization (CE-ESI)-MS. Both α-Gal- and Neu5Gc-containing N-glycans were present in all samples, with α-Gal-modified N-glycans being the most abundant nonhuman carbohydrate motif. The abundance of N-glycans terminating with α-Gal or Neu5Gc was higher in heart and kidney samples than livers. MSI revealed kidneys had the highest glycosylation levels, and α-Gal-containing N-glycans were abundant in the kidney cortex but scarce in the medulla. This study enhances our understanding of α-Gal- and Neu5Gc-modified N-glycans in animal organs and may guide research on carbohydrate antigen-induced immune rejection in xenotransplantation.