title: Structural characterization by multistage mass spectrometry (MSn) of human milk glycans recognized by human rotaviruses.
abstract: We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.(2), describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.
authors.affiliation: From the ‡Glycomics Center, University of New Hampshire, Durham, NH 03824;
authors.forename: David J
authors.initials: DJ
authors.lastname: Ashline
authors.affiliation: §Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, GA;
authors.forename: Ying
authors.initials: Y
authors.lastname: Yu
authors.affiliation: §Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, GA;
authors.forename: Yi
authors.initials: Y
authors.lastname: Lasanajak
authors.affiliation: §Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, GA;
authors.forename: Xuezheng
authors.initials: X
authors.lastname: Song
authors.affiliation: ¶Verna and Marrs McLean Department of Biochemistry and Molecular Biology;
authors.forename: Liya
authors.initials: L
authors.lastname: Hu
authors.affiliation: ‖Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX.
authors.forename: Sasirekha
authors.initials: S
authors.lastname: Ramani
authors.affiliation: ¶Verna and Marrs McLean Department of Biochemistry and Molecular Biology; ‖Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX.
authors.forename: Venkataram
authors.initials: V
authors.lastname: Prasad
authors.affiliation: ‖Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Estes
authors.affiliation: §Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, GA; vnr@unh.edu dfsmith@emory.edu rdcummi@emory.edu.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
authors.affiliation: §Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, GA; vnr@unh.edu dfsmith@emory.edu rdcummi@emory.edu.
authors.forename: David F
authors.initials: DF
authors.lastname: Smith
authors.affiliation: From the ‡Glycomics Center, University of New Hampshire, Durham, NH 03824; vnr@unh.edu dfsmith@emory.edu rdcummi@emory.edu.
authors.forename: Vernon N
authors.initials: VN
authors.lastname: Reinhold
mesh_terms: D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D054794:Glycomics; D006801:Humans; D008895:Milk, Human / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism* / Q000648:ultrastructure*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism; D012401:Rotavirus / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013194:Staining and Labeling; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D036022:Capsid Proteins; D011134:Polysaccharides; D016601:RNA-Binding Proteins; D011991:Receptors, Virus; C060339:VP4 protein, Rotavirus; D017361:Viral Nonstructural Proteins; C079234:NS35 protein, rotavirus
keywords: Human milk glycans; MSn; mass spectrometry; permethylation