Mass spectrometry for profiling LOS and lipid A structures from whole-cell lysates: Directly from a few bacterial colonies or from liquid broth cultures

Béla Kocsis, Anikó Kilár, Szandra Péter, Ágnes Dörnyei, Viktor Sándor, F. Kilár

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

Lipopolysaccharides (LPSs, endotoxins) are components of the outer cell membrane of most Gram-negative bacteria and can play an important role in a number of diseases of bacteria, including Gram-negative sepsis. The hydrophilic carbohydrate part of LPSs consists of a core oligosaccharide (in the case of an R-type LPS or lipooligosaccharide, LOS) linked to an O-polysaccharide chain (in the case of an S-type LPS), which is responsible for O-specific immunogenicity. The hydrophobic lipid A anchor is composed of a phosphorylated diglucosamine backbone to which varying numbers of ester- and amide-linked fatty acids are attached and this part of the LPSs is associated with endotoxicity. The detailed chemical characterization of endotoxins requires long-lasting large-scale isolation procedures, by which high-purity LPSs can be obtained. However, when a large number of bacterial samples and their LPS content are to be compared prompt, small-scale isolation methods are used for the preparation of endotoxins directly from bacterial cell cultures. The purity of the endotoxins extracted by these methods may not be high, but it is sufficient for analysis. Here, we describe a fast and easy micromethod suitable for extracting small quantities of LOS and a slightly modified micromethod for the detection of the lipid A constituents of the LPSs from bacteria grown in different culture media and evaluate the structures with mass spectrometry. The cellular LOS and lipid A were obtained from crude isolates of heat-killed cells, which were then subjected to matrix-assisted laser desorption/ionization mass spectrometry analysis. The observed ions in the 10-colony samples were similar to those detected for purified samples. The total time for the sample preparation and the MS analysis is less than 3 h.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages187-198
Number of pages12
DOIs
Publication statusPublished - Jan 1 2017

Publication series

NameMethods in Molecular Biology
Volume1600
ISSN (Print)1064-3745

Fingerprint

Lipid A
Endotoxins
Mass Spectrometry
Gram-Negative Bacteria
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Oligosaccharides
Amides
Polysaccharides
Culture Media
Lipopolysaccharides
Sepsis
Esters
Fatty Acids
Cell Culture Techniques
Hot Temperature
Carbohydrates
Cell Membrane
Ions
Bacteria

Keywords

  • Crude cell lysate
  • Lipid A isolation
  • LOS
  • Matrix-assisted laser desorption/ionization mass spectrometry
  • Microextraction

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Kocsis, B., Kilár, A., Péter, S., Dörnyei, Á., Sándor, V., & Kilár, F. (2017). Mass spectrometry for profiling LOS and lipid A structures from whole-cell lysates: Directly from a few bacterial colonies or from liquid broth cultures. In Methods in Molecular Biology (pp. 187-198). (Methods in Molecular Biology; Vol. 1600). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6958-6_17

Mass spectrometry for profiling LOS and lipid A structures from whole-cell lysates : Directly from a few bacterial colonies or from liquid broth cultures. / Kocsis, Béla; Kilár, Anikó; Péter, Szandra; Dörnyei, Ágnes; Sándor, Viktor; Kilár, F.

Methods in Molecular Biology. Humana Press Inc., 2017. p. 187-198 (Methods in Molecular Biology; Vol. 1600).

Research output: Chapter in Book/Report/Conference proceedingChapter

Kocsis, B, Kilár, A, Péter, S, Dörnyei, Á, Sándor, V & Kilár, F 2017, Mass spectrometry for profiling LOS and lipid A structures from whole-cell lysates: Directly from a few bacterial colonies or from liquid broth cultures. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1600, Humana Press Inc., pp. 187-198. https://doi.org/10.1007/978-1-4939-6958-6_17
Kocsis B, Kilár A, Péter S, Dörnyei Á, Sándor V, Kilár F. Mass spectrometry for profiling LOS and lipid A structures from whole-cell lysates: Directly from a few bacterial colonies or from liquid broth cultures. In Methods in Molecular Biology. Humana Press Inc. 2017. p. 187-198. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-6958-6_17
Kocsis, Béla ; Kilár, Anikó ; Péter, Szandra ; Dörnyei, Ágnes ; Sándor, Viktor ; Kilár, F. / Mass spectrometry for profiling LOS and lipid A structures from whole-cell lysates : Directly from a few bacterial colonies or from liquid broth cultures. Methods in Molecular Biology. Humana Press Inc., 2017. pp. 187-198 (Methods in Molecular Biology).
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