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Title: Structural characterization of human milk oligosaccharides using ultrahigh performance liquid chromatography–helium charge transfer dissociation mass spectrometry
Abstract The combination of helium charge transfer dissociation mass spectrometry (He–CTD–MS) with ultrahigh performance liquid chromatography (UHPLC) is presented for the analysis of a complex mixture of acidic and neutral human milk oligosaccharides (HMOs). The research focuses on the identification of the monosaccharide sequence, the branching patterns, the sialylation/fucosylation arrangements, and the differentiation of isomeric oligosaccharides in the mixture. Initial studies first optimized the conditions for the UHPLC separation and the He–CTD–MS conditions. Results demonstrate that He–CTD is compatible with UHPLC timescales and provides unambiguous glycosidic and cross-ring cleavages from both the reducing and the nonreducing ends, which is not typically possible using collision-induced dissociation. He–CTD produces informative fragments, including 0,3An and 0,4An ions, which have been observed with electron transfer dissociation, electron detachment dissociation, and ultraviolet photodissociation (UVPD) and are crucial for differentiating the α-2,3- versus α-2,6-linked sialic acid (Neu5Ac) residues present among sialyllacto-N-tetraose HMOs. In addition to the linkage positions, He–CTD is able to differentiate structural isomers for both sialyllacto-N-tetraoses and lacto-N-fucopentaoses structures by providing unique, unambiguous cross-ring cleavages of types 0,2An, 0,2Xn, and 1,5An while preserving most of the labile Neu5Ac and fucose groups.
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483 to 495
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National Science Foundation
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  1. Abstract

    Alkali and alkaline earth metal adducts of a branched glycan, XXXG, were analyzed with helium charge transfer dissociation (He‐CTD) and low‐energy collision‐induced dissociation (LE‐CID) to investigate if metalation would impact the type of fragments generated and the structural characterization of the analyte. The studied adducts included 1+ and 2+ precursors involving one or more of the cations: H+, Na+, K+, Ca2+, and Mg2+. Regardless of the metal adduct, He‐CTD generated abundant and numerous glycosidic and cross‐ring cleavages that were structurally informative and able to identify the 1,4‐linkage and 1,6‐branching patterns. In contrast, the LE‐CID spectra mainly contained glycosidic cleavages, consecutive fragments, and numerous neutral losses, which complicated spectral interpretation. LE‐CID of [M + K + H]2+and [M + Na]+precursors generated a few cross‐ring cleavages, but they were not sufficient to identify the 1,4‐linkage and 1,6‐branching pattern of the XXXG xyloglucan. He‐CTD predominantly generated 1+ fragments from 1+ precursors and 2+ product ions from 2+ precursors, although both LE‐CID and He‐CTD were able to generate 1+ product ions from 2+ adducts of magnesium and calcium. The singly charged fragments derive from the loss of H+from the metalated product ions and the formation of a protonated complementary product ion; such observations are similar to previous reports formore »magnesium and calcium salts undergoing electron capture dissociation (ECD) activation. However, during He‐CTD, the [M + Mg]2+precursor generated more singly charged product ions than [M + Ca]2+, either because Mg has a higher second ionization potential than Ca or because of conformational differences and the locations of the charging adducts during fragmentation. He‐CTD of the [M + 2Na]2+and the [M + 2 K]2+precursors generated singly charged product ions from the loss of a sodium ion and potassium ion, respectively. In summary, although the metal ions influence the mass and charge state of the observed product ions, the metal ions had a negligible effect on the types of cross‐ring cleavages observed.

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  2. Rationale

    The function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa, so the ability to identify the correct isomer using mass spectrometry can help resolve important biological questions. Tandem mass spectrometry approaches for Leu/Ile (Xle) discrimination have been developed, but they all have certain limitations.


    Four model peptides and two wild‐type peptide sequences containing either Leu or Ile residues were subjected to charge transfer dissociation (CTD) mass spectrometry on a modified three‐dimensional ion trap. The peptides were analyzed in both the 1+ and 2+ charge states, and the results were compared to conventional collision‐induced dissociation spectra of the same peptides obtained using the same instrument.


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    CTD has the benefit of being applicable to both 1+ and 2+ precursor ions, and themore »overall performance is comparable to that of other high‐energy activation techniques like hot electron capture dissociation and UV photodissociation. CTD does not require chemical modifications of the precursor peptides, nor does it require additional levels of isolation and fragmentation.

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  3. Abstract

    Glycosaminoglycans (GAGs) are linear polysaccharides found in a variety of organisms. GAGs contribute to biochemical pathway regulation, cell signaling, and disease progression. GAG sequence information is imperative for determining structure‐function relationships. Recent advances in electron‐activation techniques paired with high‐resolution mass spectrometry allow for full sequencing of GAG structures. Electron detachment dissociation (EDD) and negative electron transfer dissociation (NETD) are two electron‐activation methods that have been utilized for GAG characterization. Both methods produce an abundance of informative glycosidic and cross‐ring fragment ions without producing a high degree of sulfate decomposition. Here, we provide detailed protocols for using EDD and NETD to sequence GAG chains. In addition to protocols directly involving performing these MS/MS methods, protocols include sample preparation, method development, internal calibration, and data analysis. © 2021 Wiley Periodicals LLC.

    This article was corrected on 27 July 2022. See the end of the full text for details.

    Basic Protocol 1: Preparation of glycosaminoglycan samples

    Basic Protocol 2: FTICR method development

    Basic Protocol 3: Internal calibration with NaTFA

    Basic Protocol 4: Electron Detachment Dissociation (EDD) of GAG samples

    Basic Protocol 5: Negative electron transfer dissociation (NETD) of GAG samples

    Basic Protocol 6: Analysis of MS/MS data

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