Differentiation and classification of user-specified bacterial groups by in situ thermal hydrolysis and methylation of whole bacterial cells with tert-butyl bromide chemical ionization ion trap mass spectrometry

Abstract

Fatty acid methyl esters (FAMEs) from 20 bacterial samples, formed through in situ thermal hydrolysis and methylation (THM) of lipids from whole bacterial cells using tetramethylammonium hydroxide, have been investigated by t-butyl bromide chemical ionization (CI) ion trap mass spectrometry (MS). The mass spectra, in the early portion of the total ion profile, contained as major peaks the protonated saturated and unsaturated FAME molecular ions, along with the fragment ions, [MH−32]+ and [MH−50]+, from the monoenoic FAME isomers. The data set was tested with several multivariate statistical approaches. Gram-type classification was made based on the ratio of saturated fatty acids (mainly C15:0) to monoenoic fatty acids (mainly C18:1, C16:1, and cyclopropyl fatty acids, C17:0 and C19:0). A multivariate rule building expert system (MuRES) was used to classify targeted bacteria comprising five different genera. Cross validation of the data set showed no false classifications of the targeted organisms. Misclassifications of untargeted bacteria as target organisms were low (1.3%). The mass spectral analysis time was less than 5min per sample.