Multiple-stage precursor ion separation and high resolution mass spectrometry toward structural characterization of 2,3-diacyltrehalose family from mycobacterium tuberculosis

Mass spectrometry (MS)-based precursor ion isolation, collision-induced dissociation (CID) fragmentation, and detection using linear ion-trap multiple-stage mass spectrometry (LIT MSⁿ) in combination with high resolution mass spectrometry (HRMS) provides a unique tool for structural characterization of complex mixture without chromatographic separation. This approach permits not only separation of various lipid families and their subfamilies, but also stereoisomers, thereby, revealing the structural details. In this report, we describe the LIT MSⁿ approach to unveil the structures of a 2,3-diacyl trehalose (DAT) family isolated from the cell envelope of Mycobacterium tuberculosis, in which more than 30 molecular species, and each species consisting of up to six isomeric structures were found. LIT MSⁿ performed on both [M + Na]⁺ and [M + HCO₂]⁻ ions of DAT yield complimentary structural information for near complete characterization of the molecules, including the location of the fatty acyl substituents on the trehalose backbone. This latter information is based on the findings of the differential losses of the two fatty acyl chains in the MS² and MS³ spectra; while the product ion spectra from higher stage LIT MSⁿ permit confirmation of the structural assignment.