Use of Capillary Zone Electrophoresis Coupled to Electrospray Mass Spectrometry for the Detection and Absolute Quantitation of Peptidoglycan-Derived Peptides in Bacterial Cytoplasmic Extracts

Abstract

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%.

Recent developments in capillary and microchip electroseparations of peptides (2019–mid 2021)

“…CZE‐ESI‐MS using a 90 cm long, 50/365 μm id/od bare FS capillary, and 100 mM FA as BGE allowed baseline separation and the absolute quantification of peptidoglycan cytoplasmatic peptides (tripeptide‐pentapeptide) in the raw cytoplasmatic extract of bacteria ( Bacillus subtilis ) grown in the presence or absence of a beta‐lactam antibiotic (cephalosporin C) [230]. The absolute quantitation of the tripeptide was based on the [13C, 15N] isotopically labeled standard.…”

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

“…CZE–ESI–MS setup has been applied for the determination of three peptidoglycan‐derived peptides (tri‐, tetra‐, and pentapeptides) and their amidated derivatives in the extracts of bacterial cytoplasma [240]. These peptide fragments are generated by the bacterial autolysis of peptidoglycans—the relevant constituents of the bacterial cell wall.…”

Recent advances (2019–2021) of capillary electrophoresis‐mass spectrometry for multilevel proteomics

“…CZE‐MS/MS has been frequently used for peptidomic studies for disease‐related biomarker discovery, as shown in Section 3.1.3. Besides that, it has also been broadly used for the characterization of peptides in basic research (DeLaney & Li, 2019; Delvaux et al, 2020, 2021; Lamp et al, 2020; Liu et al, 2020; Lombard‐Banek, Yu et al, 2019; Ozawa et al, 2020; Piestansky et al, 2020).…”

“…CZE-MS/MS has been frequently used for peptidomic studies for disease-related biomarker discovery, as shown in Section 3.1.3. Besides that, it has also been broadly used for the characterization of peptides in basic research (DeLaney & Li, 2019;Delvaux et al, 2020Delvaux et al, , 2021Lamp et al, 2020;Liu et al, 2020;Lombard-Banek, Yu et al, 2019;Ozawa et al, 2020;Piestansky et al, 2020). Delvaux et al (2020) employed CZE-ESI-MS for the characterization of cysteine connectivity of disulfide bonds on synthetic and biologically relevant peptides via combinations with ion mobility-MS and theoretical calculations.…”