No abstract

The isomer ratio determination of a selenium-containing metabolite produced by Se-rich yeast was performed. Electrospray ionization and ion mobility mass spectrometry (IM-MS) were unsuccessfully used in order to resolve the isomers according to their collisional cross section (CCS) difference. The isomer ratio determination of 2,3-dihydroxypropionylselenocystathionine was performed after multidimensional liquid chromatography preconcentration from a water extract of Se-rich yeast using preparative size exclusion, anion exchange, and capillary reverse phase columns coupled to IM-MS. 4'-nitrobenzo-15-crown-5 ether, a selective shift reagent (SSR), was added after the last chromatographic dimension in order to specifically increase the CCS of one of the isomers by the formation of a stable host-guest system with the crown ether. Both isomers were consequently fully resolved by IM-MS, and the relative ratio of the isomers was determined to be 11-13% and 87-89%. The present data compared favorably with the literature to support the analytical strategy despite the lack of an authentic standard for method validation. In addition, computational chemistry methods were successfully applied to design the SSR and to support the experimental data.

Disulfide bonds between cysteine residues are commonly involved in the stability of numerous peptides and proteins and are crucial for providing biological activities. In such peptides, the appropriate cysteine connectivity ensures the proper conformation allowing an efficient binding to their molecular targets. Disulfide bond connectivity characterization is still challenging and is a critical issue in the analysis of structured peptides/proteins targeting pharmaceutical or pharmacological utilizations. This study describes the development of new and fast gas-phase and in-solution electrophoretic methods coupled to mass spectrometry to characterize the cysteine connectivity of disulfide bonds. For this purpose, disulfide isomers of three peptides bearing two intramolecular disulfide bonds but different cysteine connectivity have been investigated. Capillary zone electrophoresis and ion mobility both coupled to mass spectrometry were used to perform the separation in both aqueous and gas phases, respectively. The separation efficiency of each technique has been critically evaluated and compared. Finally, theoretical calculations were performed to support and explain the experimental data based on the predicted physicochemical properties of the different peptides.

DOI: https://doi.org/10.1002/elps.201900147 The cover picture shows the hyphenation of CE‐MS for the characterization of peptidoglycan (PGN) fragments obtained after mutanolysin digestion. CE‐MS data were favorably compared to the reverse‐phase HPLC benchmark method. Both methods provided comparable data although HPLC eluted PGN fragments of higher molecular weights. Remarkably, CE‐MS baseline resolved the modified PGN fragments containing chemical modifications of interest, namely the amidation and deacetylation.

Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and division. The resulting PGN fragments (muropeptides and peptides), which are generated by the bacterial autolytic system, are usually transported into the cytoplasm to be recycled. On the other hand, PGN fragments can act as messenger molecules involved in the bacterial cell wall stress response as in the case of β-lactamase induction in the presence of β-lactam antibiotic or in triggering mammalian innate immune response. During their cellular life, bacteria modulate their PGN degradation by their autolytic system or their recognition by the mammalian innate immune system by chemically modifying their PGN. Among these modifications, the amidation of the ε-carboxyl group of meso-diaminopimelic acid present in the PGN peptide chain is frequently observed. Currently, the detection and quantitation of PGN-derived peptides is still challenging because of the difficulty in separating these highly hydrophilic molecules by RP-HPLC as these compounds are eluted closely after the column void volume or coeluted in many cases. Here, we report the use of capillary zone electrophoresis coupled via an electrospray-based CE−MS interface to high-resolution mass spectrometry for the quantitation of three PGN peptides of interest and their amidated derivatives in bacterial cytoplasmic extracts. The absolute quantitation of the tripeptide based on the [ 13 C, 15 N] isotopically labeled standard was also performed in crude cytoplasmic extracts of bacteria grown in the presence or absence of a β-lactam antibiotic (cephalosporin C). Despite the high complexity of the samples, the repeatability of the CZE−MS quantitation results was excellent, with relative standard deviations close to 1%. The global reproducibility of the method including biological handling was better than 20%.