## 2 Results and Discussion
### Release of N-glycans from Bovine whey Proteins  
Cheese whey, a co-product of the dairy industry, is a source of \(N\)-linked glycoproteins including lactoferrin, lactoperoxidase, transferrin, and various immunoglobulins [12]. Free and immobilized Endo BI-1 was incubated with whey proteins purified from bovine colostrum at pH 7 at 45 \({}^{\circ}\)C for 1 h. The glycans were purified and characterized by nano LC-Chip Q-ToF mass spectrometry and each compound's relative abundance was determined and further classified based on the class of glycan to which each compound belongs (Figure 7). Overall, twenty-six unique \(N\)-glycan structures were identified and confirmed by tandem mass spectra. The \(N\)-glycans were characterized based on their monosaccharide composition, which included neutral (containing only hexose and \(N\)-acetylhexosamine residues), sialylated, fucosylated, and fucosylated and sialylated. For neutral glycans the immobilized enzyme released eight times higher relative amounts than free Endo BI-1. For the class fucosylated and sialylated, the free enzyme released significantly higher amount of glycans (approximately double) compared with the free enzyme. Free enzyme released 50% more of each fucosylated and sialylated glycan class. For glycans with both sialylation and fucosylation, the free enzyme released nearly double the relative amount of glycans compared with immobilization. It is worth mentioning that fucosylated and sialylated glycans were present in lowest amounts. Both sialylated and neutral glycans represented the most abundant glycan types released from colostrum whey proteins. Importantly, unique glycan compositions should only be compared between samples, and not among the various \(N\)-glycan structures themselves. In fact, differences in ionization efficiency due to diverse monosaccharide composition hinder direct comparisons in the absence of identical pure standards. The \(N\)-glycan structures released by Endo BI-1 were tabulated according to the glycan composition notation Hex_HexNAc_Fuc_NeuAc_NeuGc, which correspond with the number of hexose, \(N\)-acetylhexosamine, fucose, \(N\)-acetylheuraminic acid, and \(N\)-glycolyheuraminic acid residues respectively (Table S1). The most abundant glycans released from colostrum whey proteins by free Endo BI-1 were 4\(3\)0\(1\)0 and 3\(3\)0\(1\)0 while the most abundant glycan structures released by immobilized Endo BI-1 were 3\(3\)0\(0\)0 and 5\(1\)0\(0\)0 (Man5). The relative abundance for released  
Figure 6: Mean relative activity of immobilized Endo BI-1 on RNase B following several reuse cycles. Error bars represent one standard deviation. 3\(3\)0\(0\)0 and 5\(1\)0\(0\)0 for immobilized enzyme compared with free enzyme were 27 and 14 times higher, respectively. These large differences are the main drivers of the substantial differences in relative abundance of neutral glycans. Both 3\(3\)0\(0\)0 and 4\(3\)0\(1\)0 were identified as the most abundant glycans released from a similar protein source using Endo BI-1 [41]. Although high mannose compounds like 5\(1\)0\(0\)0 and 6\(1\)0\(0\)0 were previously found in the bovine and human milk \(N\)-glycome, likely deriving from bovine lactoferrin, they were not previously described as being released from bovine colostrum whey proteins with free Endo BI-1 [41; 43]. Despite the many technological breakthroughs, deglycosylation is still not a well-characterized process. Recently, the kinetics of the commercial enzyme peptide-\(N\)-glycosidase F (PNGase F) were investigated to understand the impact of sialylation on the release of \(N\)-glycans [44]. Presence of sialic acid slowed glycan release, while fucosylation also affected the deglycosylation rate. Because RNase B contains a single glycosylation site and high mannose type glycans, these differences would not be observed. However, when using a different enzyme (Endo BI-1) and complex substrates like bovine colostrum whey proteins under different conditions, kinetics and deglycosylation behavior would likely be substantially different.