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1. Charge transfer dissociation of a branched glycan with alkali and alkaline earth metal adducts

   https://doi.org/10.1002/jms.4774  

   Sasiene, Zachary J. ; Ropartz, David ; Rogniaux, Hélène ; Jackson, Glen P. ( June 2021 , Journal of Mass Spectrometry)

   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 for 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. Differentiation of leucine and isoleucine residues in peptides using charge transfer dissociation mass spectrometry (CTD‐MS)

   https://doi.org/10.1002/rcm.9246  

   Edwards, Halle M. ; Wu, Hoi‐Ting ; Julian, Ryan R. ; Jackson, Glen P. ( January 2022 , Rapid Communications in Mass Spectrometry)

   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.

   CTD resulted in 100% sequence coverage for each of the studied peptides and provided a variety of side‐chain cleavages, includingd,wandvions. Using CTD, reliabledandwions of Xle residues were observed more than 80% of the time. When present,dions are typically greater than 10% of the abundance of the correspondingaions from which they derive, andwions are typically more abundant than thezions from which they derive.

   CTD has the benefit of being applicable to both 1+ and 2+ precursor ions, and the 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. Electron‐Activated Tandem Mass Spectrometry Analysis of Glycosaminoglycans

   https://doi.org/10.1002/cpz1.83  

   Pepi, Lauren E. ; Amster, I. Jonathan ( April 2021 , Current Protocols)

   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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4. Substrate and Process Engineering for Biocatalytic Synthesis and Facile Purification of Human Milk Oligosaccharides

   https://doi.org/10.1002/cssc.202102539  

   Bai, Yuanyuan ; Yang, Xiaohong ; Yu, Hai ; Chen, Xi ( February 2022 , ChemSusChem)

   Innovation in process development is essential for applying biocatalysis in industrial and laboratory production of organic compounds, including beneficial carbohydrates such as human milk oligosaccharides (HMOs). HMOs have attracted increasing attention for their potential application as key ingredients in products that can improve human health. To efficiently access HMOs through biocatalysis, a combined substrate and process engineering strategy is developed, namely multistep one‐pot multienzyme (MSOPME) design. The strategy allows access to a pure tagged HMO in a single reactor with a single C18‐cartridge purification process, despite the length of the target. Its efficiency is demonstrated in the high‐yielding (71–91 %) one‐pot synthesis of twenty tagged HMOs (83–155 mg), including long‐chain oligosaccharides with or without fucosylation or sialylation up to nonaoses from a lactoside without the isolation of the intermediate oligosaccharides. Gram‐scale synthesis of an important HMO derivative – tagged lacto‐N‐fucopentaose‐I (LNFP‐I) – proceeds in 84 % yield. Tag removal is carried out in high efficiency (94–97 %) without the need for column purification to produce the desired natural HMOs with a free reducing end. The method can be readily adapted for large‐scale synthesis and automation to allow quick access to HMOs, other glycans, and glycoconjugates.

    

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5. A Solution to Antifolate Resistance in Group B Streptococcus : Untargeted Metabolomics Identifies Human Milk Oligosaccharide-Induced Perturbations That Result in Potentiation of Trimethoprim

   https://doi.org/10.1128/mBio.00076-20  

   Chambers, Schuyler A. ; Moore, Rebecca E. ; Craft, Kelly M. ; Thomas, Harrison C. ; Das, Rishub ; Manning, Shannon D. ; Codreanu, Simona G. ; Sherrod, Stacy D. ; Aronoff, David M. ; McLean, John A. ; et al ( April 2020 , mBio)

   ABSTRACT Adjuvants can be used to potentiate the function of antibiotics whose efficacy has been reduced by acquired or intrinsic resistance. In the present study, we discovered that human milk oligosaccharides (HMOs) sensitize strains of group B Streptococcus (GBS) to trimethoprim (TMP), an antibiotic to which GBS is intrinsically resistant. Reductions in the MIC of TMP reached as high as 512-fold across a diverse panel of isolates. To better understand HMOs’ mechanism of action, we characterized the metabolic response of GBS to HMO treatment using ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HRMS/MS) analysis. These data showed that when challenged by HMOs, GBS undergoes significant perturbations in metabolic pathways related to the biosynthesis and incorporation of macromolecules involved in membrane construction. This study represents reports the metabolic characterization of a cell that is perturbed by HMOs. IMPORTANCE Group B Streptococcus is an important human pathogen that causes serious infections during pregnancy which can lead to chorioamnionitis, funisitis, premature rupture of gestational membranes, preterm birth, neonatal sepsis, and death. GBS is evolving antimicrobial resistance mechanisms, and the work presented in this paper provides evidence that prebiotics such as human milk oligosaccharides can act as adjuvants to restore the utility of antibiotics. 

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