**1. Introduction**

Tandem mass spectrometry is a powerful tool for structural analysis of unknown molecules. Tandem mass spectrometry consists of several sequential events including formation and mass selection of the precursor ions, collision induced dissociation (CID) with inert target gas for fragment ion formation, followed by mass analysis and detection of the product-ions. For tandem mass spectrometry with quadrupole (e.g., triple quadrupole, TSQ), sector, and hybrid Q-TOF instruments, these processes occur sequentially in the separate regions of the instruments and the MS/MS process is tandem-in-space. For quadrupole ion-trap (QIT) and linear ion trap (LIT) instruments, the precursor ion selection, CID, and product-ion analysis and detection events are all executed in the ion trap in a timing

sequence manner, and the MS/MS process is tandem-in-time [1]. Linear ion trap multiple stage tandem mass spectrometry (LIT MSn) permits the repeat of the precursor ion selection-CID-product-ion analysis process, and up to 10 cycles can be performed using modern commercial LIT mass spectrometers such as Thermo LTQ Orbitrap. This instrument is also featured with an extreme high resolving power and up to a million resolution (at m/z 200) can be reached [2].

Due to its MSn capability with high resolution, LIT/Orbitrap with MSn approach has been widely used in the structural characterization of a wide range of biomolecules [3–7]. LIT MSn with high resolution is also extremely useful for identification of complex lipid structures, in particular, microbial lipid, permitting revelation of numerous isomeric molecules in lipid extracts. For example, we demonstrated that sulfolipid II in *Mycobacterium tuberculosis* (*M. tuberculosis*) H37Rv cells is the predominated lipid family, consisting of hundreds of molecular species rather than the sulfolipid I family as previously reported [8,9]. LIT MSn with high resolution mass spectrometry has also been successfully applied to delineate the structures of phosphatidylinositol mannosides (PIMs) [10,11], and phthiocerol dimycocerosates (PDIMs) [12] in the cell envelope of *M. tuberculosis*. The former lipid family is known to have played important roles in *M. tuberculosis* adhesins that mediate attachment to nonphagocytic cells [13], while the latter is recently found to play role in drug resistance to *M. tuberculosis* [14].

In addition to the above complex lipid families, other glycolipids found in the mycobacterial cell wall include acylated trehaloses [15–18]. These trehalose-containing glycolipids consist of many families [18–22], of which the 2,3-di-O-acyltrehaIose (DAT) family was previously defined as glycolipid B. DAT is a mycobacterial factor capable of modulating host immune responses [23] and can inhibit the proliferation of murine T cells [24]. DAT along with pentaacyl trehalose (PAT) also play an important role in pathogenesis and a structural role in the cell envelope, promoting the intracellular survival of the bacterium [25]. The DATs from *M. tuberculosis* and *M. fortuitum* have been shown to be antigenic [24,26,27] and their potential use in serodiagnosis has been postulated [28,29].

Besra and coworker defined the structures of the acylated trehalose lipid family using gas chromatography-mass spectrometry, in conjunction with normal/reversed phase TLC, and one/two-dimensional 1H, and 13C nuclear magnetic resonance spectroscopy [21]. However, there is no report in the detailed structural assignment of the various molecular species with many isomeric structures for the entire lipid family.

In this report, we applied multiple stage precursor ion isolation and resonance excitation activation to generate distinct MSn spectra to explore the structure details of the 2,3-diacyl trehalose (DAT) lipid family found in *M. tuberculosis*. This study highlights the unique feature of the technique of LIT MSn for tandem-in-time precursor ion separation that affords structural characterization of a complex lipid family, while a similar structural analysis would be very difficult to perform utilizing the tandem mass spectrometric approach with a sector, TSQ or QTOF instrument.

### **2. Materials and Methods**
