Ultra-Sensitive, High-Resolution Liquid Chromatography Methods for the High-Throughput Quantitative Analysis of Bacterial Cell Wall Chemistry and Structure.
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.
Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Centre for Microbial Research, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 90187, Sweden.
Laura
L
Alvarez
Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Centre for Microbial Research, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 90187, Sweden.
Sara B
SB
Hernandez
Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, 28049, Spain.
Miguel A
MA
de Pedro
Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Centre for Microbial Research, Umeå University, 6K och 6L, Sjukhusområdet, Umeå, 90187, Sweden. felipe.cava@umu.se.
Felipe
F
Cava
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D006090:Gram-Negative Bacteria / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D010457:Peptidoglycan / Q000737:chemistry / Q000302:isolation & purification*; D012997:Solvents; D013997:Time Factors
D001426:Bacterial Proteins; D010457:Peptidoglycan; D012997:Solvents
Cell wall; HPLC; Muropeptide; Peptidoglycan; Reverse-phase liquid chromatography; UPLC