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.