The Multiplicity of N-Glycan Structures of Bovine Milk 18 kDa Lactophorin (Milk GlyCAM-1)

Lactophorin is a heat-stable phosphoglycoprotein, also known as milk glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1). Bovine 18 kDa lactophorin was purified by heparin affinity chromatography from cow's milk whey. Its N-glycans were obtained by proteomic techniques, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by in-gel digestion with peptide-N-4-(N-acetyl-beta-glucosaminyl)-asparagine amidase (PNGase F). The released N-glycans were derivatized with 2-aminopryridine (PA.) and analyzed by matrix-assisted laser desorption ionization quadruple ion trap time of flight mass spectrometry (MALDI-QIT-TOF MS). Among the MS analyzed peaks, 15 peaks were found to be N-glycan molecules as detected by MS2 analysis. These glycans consisted of mono-sialylated bi-, tri-, and tetra-antennary complex-type N-glycans carrying Gal-GlcNAc (LacNAc) or GalNAc-GlcNAc (LacdiNAc) with and without core-fucose.