Structural definition of trehalose 6-monomycolates and trehalose 6,6'-dimycolates from the pathogen Rhodococcus equi by multiple-stage linear ion-trap mass spectrometry with electrospray ionization

Abstract

The cell wall of the pathogenic bacterium Rhodococcus equi (R. equi) contains abundant trehalose monomycolate (TMM) and trehalose dimycolate (TDM), the glycolipids bearing mycolic acids. Here, we describe multiple-stage (MS(n)) linear ion-trap (LIT) mass spectrometric approaches toward structural characterization of TMM and TDM desorbed as [M + Alk](+) (Alk = Na, Li) and as [M + X](-) (X = CH(3)CO(2), HCO(2)) ions by electrospray ionization (ESI). Upon MS(n) (n=2, 3, 4) on the [M + Alk](+) or the [M + X](-) adduct ions of TMM and TDM, abundant structurally informative fragment ions are readily available, permitting fast assignment of the length of the meromycolate chain and of the α-branch on the mycolyl residues. In this way, structures of TMM and TDM isolated from pathogenic R. equi strain 103 can be determined. Our results indicate that the major TMM and TDM molecules possess 6, and/or 6'-mycolyl groups that consist of mainly C14 and C16 α-branches with meromycolate branches ranging from C18 to C28, similar to the structures of the unbound mycolic acids found in the cell envelope. Up to 60 isobaric isomers varying in chain length of the α-branch and of the meromycolate backbone were observed for some of the TDM species in the mixture. This mass spectrometric approach provides a direct method that affords identification of various TMM and TDM isomers in a mixture of which the complexity of this lipid class has not been previously reported using other analytical methods.

Figure 1

The ESI-MS spectra of the [M + Na]⁺ ions of TMM (a) and of TDM (b). These profiles are similar to their [M + CH₃CO₂]⁻ adduct ions (see supplemental material Figure S1).

Figure 2

The LIT MS² spectrum of the [M + Na]⁺ ion of TMM at m/z 871 (a), its MS³ spectra at m/z 603 (871 → 603) (b), and at m/z 709 (871 → 709) (c).

Figure 3

The LIT MS² spectrum of the [M + Na]⁺ ion of TDM at m/z 1378 (a), its MS³ spectra at m/z 1109 (1378 → 1109) (b), its MS⁴ spectra at m/z 813 (1378 → 1109 → 813) (c), and at m/z 737 (1378 → 1109 → 737) (d); the MS³ spectra of the ions of m/z 1081 (1378 → 1081) (e), and of m/z 1053 (1378 → 1053) (f)

Figure 4

The LIT MS² spectra of the [M + CH₃CO₂]^- ion of TMM at m/z 907 (a), its MS³ spectra at m/z 829 (907 → 829) (b), and at m/z 579 (907 → 579) (c).

Scheme 1

The fragmenation pathways proposed for the [M + Na]⁺ ions of 6-mycolyl-α,α'-D-trehalose (TMM) (the indicated m/z values are ions seen for18:0/16:0-TMM, which is one of the 3 isomers that give rise to the [M+Na]⁺ of m/z 871)

Scheme 2

The fragmentation tree applying multiple-stage mass spectrometry (MSⁿ) for structural assignment of the [M + Na]⁺ ions of 6,6'-dimycolyl-α,α'-D-trehalose (TDM).

Scheme 3

The fragmentation pathways proposed for the [M + Na]⁺ ions of 6,6'-dimycolyl-α,α'-D-trehalose (TDM). The indicated m/z values are fragment ions seen for (20:0/14:0-18:0/16:0-TDM isomer, which is one of the major isomers that represent the sodiated molecular species of m/z 1378.1