Structural determination of glycopeptidolipids of Mycobacterium smegmatis by high-resolution multiple-stage linear ion-trap mass spectrometry with electrospray ionization.

Glycopeptidolipids (GPLs) are abundant in the cell walls of different species of mycobacteria and consist of tripeptide-amino-alcohol core of D-Phe-D-allo-Thr-D-Ala-L-alaninol linked to 3-hydroxy or 3-methoxy C(26-34) fatty acyl chain at the N-terminal of D-Phe via amide linkage, and a 6-deoxytalose (6-dTal) and an O-methyl rhamnose residues, respectively, attach to D-allo-Thr and the terminal L-alaninol. They are important cell-surface antigens that are implicated in the pathogenesis of opportunistic mycobacteria belonging to the Mycobacterium avium complex. In this contribution, we described multiple-stage linear ion trap in conjunction with high-resolution mass spectrometry towards structural characterization of complex GPLs as [M + Na](+) ions isolated from Mycobacterium smegmatis, a fast-growing and non-pathogenic mycobacterial species. Following resonance excitation in an ion trap, MS(n) spectra of the [M + Na](+) ions of GPLs contained mainly b and y series ions that readily determine the peptide sequence. Fragment ions from MS(n) also afford locating the 6-dTal and O-methyl rhamnose residues linked to the D-allo-Thr and terminal L-alaninol of the peptide core, respectively, as well as recognizing the modifications of the glycosides, including their acetylation and methylation states and the presence of succinyl group. The GPL families consisting of 3-hydroxy fatty acyl and of 3-methoxy fatty acyl substituents are readily distinguishable. The MS profiles of the GPLs from cells are dependant on the conditions they were grown, and several isobaric isomers were identified for many of the molecular species. These multiple-stage mass spectrometric approaches give detailed structures of GPL in complex mixtures of which the isomeric structures are difficult to define using other analytical methods.