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. Molecular & Cellular

Human DC-SIGN binds specific human milk glycans

“…The fucosylated glycan determinant present in these four structures was likely Blood Group H Type 1 (H1) since the anti-H1 antibody but none of the Lewis antibodies bound these four structures. Additionally, HMO-23, -31, -41, -47, -48, and -49, containing 1 or 2 fucoses, were also not bound and contain only an internal Lewis x determinant (or, in the case of HMO-31, internal Lewis x as the major structures) [21, 28]. Therefore, the Lewis x is a binding determinant of DC-SIGN only when present at the non-reducing end of HMGs.…”

“…All of the other lectins, antibodies, and glycosidases used for MAGS, as well as the concentration(s) and glycosidase treatment procedures, are the same as described in a previous study [21]. Multi-dimensional mass spectrometry on HMG-9, HMG-19, and HMG-36 was performed as previously described [28]. …”

“…Proposed structures for these HMGs samples are shown. HMG samples in bold-face font were further analyzed by multi-dimensional mass spectrometry (MS n ) to more accurately determine the structures(s) within these samples; HMG-20, -21, and -28 were previously sequenced by MS n [21, 28] and HMGs-9, -19, and -36 were also by sequenced by MS n in a more recent manuscript [33]. Refer to Supplementary File 2 for the lectin and antibody screening data.…”

Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing

“…Moreover, it should be valuable as a complement to orthogonal methods such as mass spectrometry and sequencing chromatography. The latter methods reveal glycan compositions and some structural information, but additional data about particular motifs could help to resolve ambiguities 48 . With further work to make the protocols routine and the reagents and software readily available, the method could improve accessibility of glycan analysis to researchers involved in a broad range of studies.…”

Divergent Chemoenzymatic Synthesis of Asymmetrical‐Core‐Fucosylated and Core‐Unmodified N‐Glycans

“…different structures with identical molecular weights), which greatly complicates biomarker discovery. [13] It is the expectation that well-defined glycan standards will make it possible to develop methodologies that can identify exact structures of glycans. [14] …”