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The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R), created by α-1,3-glycosyltransferase (GT), is recognized by natural anti-α-1,3-galactose antibodies in human serum and is responsible for hyperacute rejection (HAR) in pig-to-human/primate xenotransplantation. Although a bioxeno-organ such as heart, kidney, and islet cells from GT knockout (GT KO) pigs has overcome hyperacute rejection, an isoglobotriosylceramide synthase (iGb3s) may produce additional α-1,3-terminated galactose residues on glycosphingolipid. This study was performed to investigate the effect of additional iGb3s KO on the glycoproteomic characterization in the GT-MCP/-MCP (GT/MCP) transgenic pig cells. The GT-MCP/-MCP ear fibroblast cells were obtained from GT-MCP/-MCP pig. The transgenic cells were knocked out with iGb3s and confirmed as GT-MCP/-MCP+iGb3s-/- (GT/MCP/iGb3s) cells. WT pig cells were used as a control. To characterize the expression of α-Gal epitope, the both GT/MCP and GT/MCP/iGb3s cells were analyzed by FACS using BS-IB4 lectin antibody. The α-Gal epitope expression in both TG cells was significan3tly reduced as compared with WT (p<0.05). To analyze a change of glycoprotein levels, the conditioned medium after WT, GT/MCP, and GT/MCP/iGb3s cell culture was collected and performed LC-MS/MS analysis. The data was qualified by GPA (GlycoProteome Analyzer). A total amount of glycopeptide in GT/MCP/iGb3s was increased as compared with that of GT/MCP and WT. However, the glycopeptide forms connected Gal-Gal were reduced in GT/MCP/iGb3s compared to both WT and GT/MCP. On the other hand, the glycopeptides including fucose and sialic acid were increased in GT/MCP/iGb3s compared to both WT and GT/MCP. Our results demonstrated that additional KO of iGb3s combined with GT KO reduced the expressions of α-Gal epitope and glycopeptide forms connecting Gal-Gal. Further studies are needed to product TG piglets and evaluate synergic effects of double gene knock out in order to minimize HAR response after xenotransplantation.This work was carried out with the support of "Cooperative Research Program for Animal Science & Technology Development (Project No. PJ010956)” of Rural Development Administration, Republic of Korea.