Precision and sensitivity of the measurement of 15N enrichment in D-alanine from bacterial cell walls using positive/negative ion mass spectrometry

Abstract

Sensitive detection of cellular components from specific groups of microbes can be utilized as ‘signatures’ in the examination of microbial consortia from soils, sediments or biofilms. Utilizing capillary gas chromatography/mass spectrometry and stereospecific derivatizing agents, D-alanine, a component localized in the prokaryotic (bacteria) cell wall, can be detected reproducibly. Enrichments of D- 15N]alanine determined in E. coli grown with [ 15N]ammonia can be determined with precision at 1.0 atom%. Chemical ionization with methane gas and the detection of negative ions (M–HF) −· and (M−F or M+H − HF) − formed from the heptafluorobutyryl D-2 butanol ester of D-alanine allowed as little as 8 pg (90 fmol) to be detected reproducibly. This method can be utilized to define the metabolic activity in terms of 15N incorporation at the level of 10 3−10 4 cells, as a function of the 15N− 14N ratio.