Negative and positive ion matrix-assisted laser desorption ionization mass spectrometry of peptidoglycan fragments after size fractionation and reversed-phase high-performance liquid chromatography

Abstract

In this paper, we outline a general applicable matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS)-based strategy for the characterization of sodium borohydride-reduced peptidoglycan fragments (dimeric up to oligomeric fragments) derived from muramidase-digested murein. As an example, murein isolated from the cyanelles of the photoautotrophic Cyanophora paradoxa was selected. Separation of unreduced peptidoglycan fragments (generated by muramidase digestion) by gel filtration divided them into muropeptides according to their sizes (degree of polymerization). Afterwards, the pooled fractions were reduced by NaBH 4 and isolation of the individual peptidoglycan fragments was performed by reversed-phase high-performance liquid chromatography (RP HPLC), with a sodium- or potassium phosphate-containing water–methanol gradient giving the maximal separation efficiency. For determining the exact molecular masses of these individual components, MALDI MS proved to be the most sensitive mass spectrometric technique for these salt-containing samples. A desalting step based on a further HPLC step, using a water–acetonitrile–trifluoroacetic acid gradient turned out to be very helpful for the molecular mass determination, especially of large peptidoglycan fragments (molecular weight range >3500 Da), which were present in very small quantities, but were not absolutely necessary. In the negative ion, reflectron mode, we could achieve the best signal-to-noise ratio and a mass accuracy of −0.03 to +0.1% for deprotonated molecular ions. The combined results from negative (and positive ion) MALDI MS in the reflectron mode, gel chromatography, HPLC and amino acid/sugar analysis (on the total hydrolysate of the purified murein) allowed us to deduce the primary structure of dimeric up to tetrameric peptidoglycan fragments isolated from the cyanelle murein of C. paradoxa.