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.
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.
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%.
For decades, structural
analysis of proteins have received considerable
attention, from their sequencing to the determination of their 3D
structures either in the free state (e.g., no host–guest system,
apoproteins) or (non)covalently bound complexes. The elucidation of
the 3D structures and the mapping of intra- and intermolecular interactions
are valuable sources of information to understand the physicochemical
properties of such systems. X-ray crystallography and nuclear magnetic
resonance are methods of choice for obtaining structures at the atomic
level. Nonetheless, they still present drawbacks which limit their
use to highly purified systems in a relatively high amount. On the
contrary, mass spectrometry (MS) has become a powerful tool thanks
to its selectivity, sensitivity, and the development of structural
methods both at the global shape and the residue level. The combination
of several MS-based methods is mandatory to fully assign a putative
structure in combination with computational chemistry and bioinformatics.
In that context, we propose a strategy which complements the existing
methods of structural studies (e.g., circular dichroism, hydrogen/deuterium
exchange and cross-links experiments, nuclear magnetic resonance).
The workflow is based on the collection of structural information
on proteins from the apparition rates and the time of appearance of
released peptides generated by a protease in controlled experimental
conditions with online detection by electrospray high-resolution mass
spectrometry. Nondenaturing, partially or fully denatured proteins
were digested by the enzymatic reactor, i.e., β-lactoglobulin,
cytochrome c, and β-casein. The collected data
are interpreted with regard to the kinetic schemes with time-dependent
rates of the enzymatic digestion established beforehand, considering
kinetics parameters in the Michaelis–Menten formalism including k
cat (the turnover number), k
1 (formation of the enzyme–substrate complex), k
–1 (dissociation of the enzyme–substrate
complex), k
off (local refolding of the
protein around the cleavage site), and k
on (local unfolding of the protein around the cleavage site). Solvent-accessible
surface analysis through digestion kinetics was also investigated.
The initial apparition rates of released peptides varied according
to the protein state (folded vs denatured) and informs the k
off/k
on ratio around
the cleavage site. On the other hand, the time of appearance of a
given peptide is related to its solvent accessibility and to the resilience
of the residual protein structure in solution. Temperature-dependent
digestion experiments allowed estimation of the type of secondary
structures around the cleavage site.
Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non‐soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N‐acetyl‐muramidase, cleaving the β1→4 bound between N‐acetylglucosamine and N‐acetylmuramic acid. Here, we report the use of CZE–MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID–MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.
The research eld of process mining deals with the extraction of knowledge from event logs. Event logs consist of the recording of activities that took place in a certain business environment and as such, one of process mining's main goals is to get an insight on the execution of business processes. Although a substantial eort has been put on developing techniques which are able to mine event logs accurately, it is still unclear how exactly characteristics of the latter inuence a technique's performance. In this paper, we provide a robust methodology of analysis and subsequently derive useful insights on the role of event log characteristics in process discovery tasks by means of an exhaustive comparative study.
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