ABSTRACT
As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and ß-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and ß-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and ß-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N/O-glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O-glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N-glycosylation modification, and the ß-subunit only O-glycosylation, a total of 22 N-glycans and 20 O-glycans were identified in the α- and ß-subunit, respectively. In addition, the complex N-glycan (47 %) and the sialylated O-glycan (77 %) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans.
Subject(s)
Chickens , Electrophoresis, Polyacrylamide Gel , Ovomucin , Polysaccharides , Animals , Glycosylation , Liquid Chromatography-Mass Spectrometry , Ovomucin/chemistry , Polysaccharides/chemistry , Polysaccharides/analysis , Protein Subunits/chemistryABSTRACT
Glycans mediate various biological processes through carbohydrate-protein interactions, and glycan microarrays have become indispensable tools for understanding these mechanisms. However, advances in functional glycomics are hindered by the absence of convenient and universal methods for obtaining natural glycan libraries with diverse structures from glycoconjugates. To address this challenge, we have developed an integrative approach that enables one-pot release and simultaneously capture, separation, structural characterization, and functional analysis of N/O-glycans. Using this approach, glycoconjugates are incubated with a pyrazolone-type heterobifunctional tag-ANPMP to obtain glycan-2ANPMP conjugates, which are then converted to glycan-AEPMP conjugates. We prepared a tagged glycan library from porcine gastric mucin, soy protein, human milk oligosaccharides, etc. Following derivatization by N-acetylation and permethylation, glycans were subjected to detailed structural characterization by ESI-MSn analysis, which revealed >83 highly pure glycan-AEPMPs containing various natural glycan epitopes. A shotgun microarray is constructed to study the fine details of glycan-bindings by proteins and antisera.
Subject(s)
Proteins , Pyrazolones , Animals , Humans , Swine , Glycoconjugates , Polysaccharides/chemistry , Glycomics/methodsABSTRACT
Glycosphingolipids participate in brain development, intestinal tract maturation, and defense against gut pathogens. Here, we performed a qualitative and quantitative comparison of milk glycosphingolipids from secretors and nonsecretors. Hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry was employed, along with an internal standard, to resolve the complications presented by the fact that glycosphingolipids are structurally diverse, varying in glycan composition and ceramide. In total, 101 glycosphingolipids were detected, of which 76 were reported for the first time, including fucose-modified neutral glycosphingolipids. Seventy-eight glycosphingolipids differed significantly between secretor and nonsecretor milk (p < 0.05), resulting in higher levels of certain neutral species (p < 0.001) but lower levels of fucose-modified monosialylated and disialylated species in secretor mothers (p < 0.01). In both milk types, the most abundant glycosphingolipids were of the monosialylated type, followed by disialylated, neutral, and trisialylated ones. Notably, fucose-modified monosialylated glycosphingolipids accounted for the highest proportion.
Subject(s)
Milk, Human , Tandem Mass Spectrometry , Female , Humans , Milk, Human/chemistry , Fucose , Glycosphingolipids/chemistry , Mothers , Oligosaccharides/chemistryABSTRACT
Human milk glycans are complex carbohydrates, which play a pivotal role in infant health and neonatal development. Maternal secretor status is known to affect free oligosaccharides in milk. Here, the milk N-glycome of secretor (Se+) and nonsecretor (Se-) individuals was qualitatively and quantitatively analyzed by hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry. The total glycosylation, fucosylation, and sialylation of N-glycans was three times higher in the Se+ group compared to the Se- group (p < 0.001) per equal volume of milk. Importantly, 52 out of 63 N-glycans-including the eight most abundant ones-differed greatly between Se+ and Se- individuals (p < 0.05). Moreover, nine N-glycans (H5N3F1, H6N3, H3N5F1, H5N5F1, H5N5F1S1, H5N4F3S1, H6N4F2S1, H6N5F4S1, and H8N7S1) were >10 times more abundant in Se+ milk than in Se- milk. These findings lay a glycomics-basis for designing personalized nutrition supplements for infants.
Subject(s)
Milk, Human , Whey , Infant, Newborn , Humans , Milk, Human/chemistry , Whey/chemistry , Polysaccharides/chemistry , Oligosaccharides/chemistry , Whey Proteins , GlycoproteinsABSTRACT
The structure of chondroitin sulfate oligosaccharides (CSOs), especially their sulfation pattern, has been found to be closely related with many biological pathways and diseases. However, detailed functional analysis such as their interaction with glycan binding proteins (GBPs) has been lagging, presumably due to the unavailability of well-defined, diverse structures. Besides challenging chemical and enzymatic synthesis, this is also due to the challenges in their purification at the isomer level and structural analysis owing to their instability, structural complexity, and low mass spectrometry detection sensitivity. Herein, we first used recycling preparative HPLC to separate and purify shark CS tetrasaccharide component labeled by a bifunctional fluorescent linker 2-amino-N-(2-aminoethyl)benzamide (AEAB) at the isomer level. Then, each isomer was derivatized through a multistage procedure including N-acetylation, carboxyl amidation, permethylation, and desulfation with silylating reagent. Structural analysis of each derivatized isomer was performed with ESI-MSn in positive ion mode. A total of 16 isomers of CSO-AEAB were isolated, with a minimum mass component of 0.007 mg and a maximum mass component of 17.53 mg, of which 10 isomers (>90 µg) were structurally analyzed. This preparation and structure analysis of CSOs lay the foundation for further study of the structure-activity relationship of CSOs.