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Ultra-Sensitive, High-Resolution Liquid Chromatography Methods for the High-Throughput Quantitative Analysis of Bacterial Cell Wall Chemistry and Structure

  • Laura Alvarez
  • Sara B. Hernandez
  • Miguel A. de Pedro
  • Felipe CavaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1440)

Abstract

High-performance liquid chromatography (HPLC) analysis has been critical for determining the structural and chemical complexity of the cell wall. However this method is very time consuming in terms of sample preparation and chromatographic separation. Here we describe (1) optimized methods for peptidoglycan isolation from both Gram-negative and Gram-positive bacteria that dramatically reduce the sample preparation time, and (2) the application of the fast and highly efficient ultra-performance liquid chromatography (UPLC) technology to muropeptide separation and quantification. The advances in both analytical instrumentation and stationary-phase chemistry have allowed for evolved protocols which cut run time from hours (2–3 h) to minutes (10–20 min), and sample demands by at least one order of magnitude. Furthermore, development of methods based on organic solvents permits in-line mass spectrometry (MS) of the UPLC-resolved muropeptides. Application of these technologies to high-throughput analysis will expedite the better understanding of the cell wall biology.

Key words

UPLC HPLC Reverse-phase liquid chromatography Cell wall Peptidoglycan Muropeptide 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Laura Alvarez
    • 1
  • Sara B. Hernandez
    • 1
  • Miguel A. de Pedro
    • 2
  • Felipe Cava
    • 1
    Email author
  1. 1.Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Centre for Microbial ResearchUmeå UniversityUmeåSweden
  2. 2.Centro de Biología Molecular “Severo Ochoa”Universidad Autónoma de Madrid-Consejo Superior de Investigaciones CientíficasMadridSpain

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