NEGATIVE AND POSITIVE-ION MATRIX-ASSISTED LASER-DESORPTION IONIZATIONMASS-SPECTROMETRY OF PEPTIDOGLYCAN FRAGMENTS AFTER SIZE FRACTIONATIONAND REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

In this paper, we outline a general applicable matrix-assisted laser d esorption ionization (MALDI) mass spectrometry(MS)-based strategy for the characterization of sodium borohydride-reduced peptidoglycan fragm ents (dimeric up to oligomeric fragments) derived from muramidase-dige sted murein. As an example,murein isolated from the cyanelles of the p hotoautotrophic Cyanophora paradoxa was selected. Separation of unredu ced peptidoglycan fragments (generated by muramidase digestion) by gel filtration divided them into muropeptides according to their sizes (d egree of polymerization). Afterwards, the pooled fr actions were reduc ed by NaBH4 and isolation of the individual peptidoglycan fragments wa s performed by reversed-phase high-performance liquid chromatography ( RP HPLC), with a sodium- or potassium phosphate-containing water-metha nol gradient giving the maximal separation efficiency. For determining the exact molecular masses of these individual components, MALDI MS p roved 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 (moleculal- weight range >3500 Dal, whi ch were present in very small quantities, but were not absolutely nece ssary. 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 depro tonated molecular ions. The combined results from negative land positi ve ion) MALDI MS in the reflectron mode, gel chromatography, HPLC and amino acid/sugar analysis ton the total hydrolysate of the purified mu rein) allowed us to deduce the primary structure of dimeric up to tetr americ peptidoglycan fragments isolated from the cyanelle murein of C. paradoxa.. (C) 1998 Elsevier Science B.V.