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Method revealing bacterial cell-wall architecture by time-dependent isotope labeling and quantitative liquid chromatography/mass spectrometry.

ABSTRACT: The molecular details of the biosynthesis and resulting architecture of the bacterial cell wall remain unclear but are essential to understanding the activity of glycopeptide antibiotics, the recognition of pathogens by hosts, and the processes of bacterial growth and division. Here we report a new strategy to elucidate bacterial cell-wall architecture based on time-dependent isotope labeling of bacterial cells quantified by liquid chromatography/accurate mass measurement mass spectrometry. The results allow us to track the fate of cell-wall precursors (which contain the vancomycin-binding site) in Enterococcus faecium, a leading antibiotic-resistant pathogen. By comparing isotopic enrichments of postinsertionally modified cell-wall precursors, we find that tripeptides and species without aspartic acid/asparagine (Asp/Asn, Asx) bridges are specific to mature cell wall. Additionally, we find that the sequence of cell-wall maturation varies throughout a cell cycle. We suggest that actively dividing E. faecium cells have three zones of unique peptidoglycan processing. Our results reveal new organizational characteristics of the bacterial cell wall that are important to understanding tertiary structure and designing novel drugs for antibiotic-resistant pathogens.

SUBMITTER: Patti GJ

PROVIDER: S-EPMC2715431 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Method revealing bacterial cell-wall architecture by time-dependent isotope labeling and quantitative liquid chromatography/mass spectrometry.

Patti Gary J GJ Chen Jiawei J Gross Michael L ML

Analytical chemistry 20090401 7

The molecular details of the biosynthesis and resulting architecture of the bacterial cell wall remain unclear but are essential to understanding the activity of glycopeptide antibiotics, the recognition of pathogens by hosts, and the processes of bacterial growth and division. Here we report a new strategy to elucidate bacterial cell-wall architecture based on time-dependent isotope labeling of bacterial cells quantified by liquid chromatography/accurate mass measurement mass spectrometry. The ...[more]

PMID: 19281243

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Project description:Circulating exosomes in bodily fluids such as blood are being actively studied as a rich source of chemical biomarkers for cancer diagnosis and monitoring. Although nucleic acid analysis is a primary tool for the discovery of circulating biomarkers in exosomes, metabolomics holds the potential of expanding the chemical diversity of biomarkers that may be easy and rapid to detect. However, only trace amounts of exosomes can be isolated from a small volume of patient blood, and thus a very sensitive technique is required to analyze the metabolome of exosomes. In this report, we present a workflow that involves multiple cycles of ultracentrifugation for exosome isolation using a starting material of 2 mL of human serum, freeze-thaw-cycles in 50% methanol/water for exosome lysis and metabolite extraction, differential chemical isotope labeling (CIL) of metabolites for enhancing liquid chromatography (LC) separation and improving mass spectrometry (MS) detection, and nanoflow LC-MS (nLC-MS) with captivespray for analysis. As a proof-of-principle, we used dansylation labeling to analyze the amine- and phenol-submetabolomes in two sets of exosome samples isolated from the blood samples of five pancreatic cancer patients before and after chemotherapy treatment. The average number of peak pairs or metabolites detected was 1964 ± 60 per sample for a total of 2446 peak pairs ( n = 10) in the first set and 1948 ± 117 per sample for a total of 2511 peak pairs ( n = 10) in the second set. There were 101 and 94 metabolites positively identified in the first and second set, respectively, and 1580 and 1590 peak pairs with accurate masses matching those of metabolites in the MyCompoundID metabolome database. Analyzing the mixtures of 12C-labeled individual exosome samples spiked with a 13C-labeled pooled sample which served as an internal standard allowed relative quantification of metabolomic changes of exosomes of blood samples collected before and after treatment.

Dataset Information

Method revealing bacterial cell-wall architecture by time-dependent isotope labeling and quantitative liquid chromatography/mass spectrometry.

Publications

Method revealing bacterial cell-wall architecture by time-dependent isotope labeling and quantitative liquid chromatography/mass spectrometry.

Similar Datasets

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