Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS

Marina Borisova; Christoph Mayer

doi:10.21769/BioProtoc.2420

Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS

Bio Protoc. 2017 Aug 5;7(15):e2420. doi: 10.21769/BioProtoc.2420.

Authors

Marina Borisova¹, Christoph Mayer¹

Affiliation

¹ Department of Microbiology and Biotechnology, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany.

Abstract

We describe here in detail a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based method to determine N-acetylmuramic acid-6-phosphate (MurNAc-6P) in bacterial cell extracts. The method can be applied to both Gram-negative and Gram-positive bacteria, and as an example we use Escherichia coli cells in this study. Wild type and mutant cells are grown for a defined time in a medium of choice and harvested by centrifugation. Then the cells are disintegrated and soluble cell extracts are generated. After removal of proteins by precipitation with acetone, the extracts are analyzed by HPLC-MS. Base peak chromatograms of wild type and mutant cell extracts are used to determine a differential ion spectrum that reveals differences in the MurNAc-6P content of the two samples. Determination of peak areas of extracted chromatograms of MurNAc-6P ((M-H)- = 372.070 m/z in negative ion mode) allows quantifying MurNAc-6P levels, that are used to calculate recycling rates of the MurNAc-content of peptidoglycan.

Keywords: Bacteria; Base peak chromatogram (BPC); Cell wall metabolism; Cytosolic metabolites; Extracted ion chromatogram (EIC); LC-MS; MurNAc-6P accumulation; Peptidoglycan recycling.