Determination of diamino acids in peptidoglycans from anaerobic bacteria

Peptidoglycans isolated from two Fusobacterium species of anaerobic bacteri a were analyzed for constituent amino acids. Hydrolysis conditions were var ied to optimize the yield of diamino acids from peptidoglycan. The o-phthal aldehyde derivatives of the diamino acid stereoisomers were separated by hi gh-performance liquid chromatography (OPA-HPLC), and variations in the rela tive areas of the two peaks noticed during analysis of solid samples were a ttributed to sampling errors. Co-derivatization/injection experiments using standards of the meso and rac forms separated from commercial mixtures dem onstrated that meso-2,6-diaminopimelic acid and meso-lanthionine were pepti doglyean components in Fusobacterium varium and Fusobacterium nucleatum, re spectively. The protonated molecules of 2,6-diaminopimelic acid and lanthio nine were detected in peptidoglycan hydrolyzates by off-line, flow-injectio n electrospray mass spectrometry (ESI-MS). In ESI-MS-MS experiments under i dentical collision-induced dissociation (CID) conditions, peptidoglycan-der ived and standard diamino acids exhibited similar fragmentations. Fragmenta tion pathways are proposed for each diamino acid. The results confirm that the meso forms of two different diamino acids are utilized in the peptidogl ycans of Fusobacterium species.