**4. Conclusions**

Sequential precursor ion isolation applying multiple-stage mass spectrometry (LIT MSn) adds another dimension of separation in the analysis, providing a powerful tool for structural identification of various compounds. Thereby, many isomeric structures of the molecule can be unveiled in a very short period of time. By contrast, using the conventional chromatographic separation combined with a TSQ or QTOF instrument, the consecutive precursor ion isolation by MS is not achievable, and the species separation can only rely on column separation. Thus, complete separation of a complex lipid mixture with a wide range of molecular species and many isomeric structures is often difficult. Compound separation by chromatographic means also requires significantly more times [33], as compared to the LIT MSn approach, by which the separation-CID-detection process can be completed within a very short period of time.

LIT MSn permits ion isolation in the time sequence manner, and the separation of ions is flexible (i.e., the types of ions selected and the mass selection window of precursor ions). The selected ions become more specific, and the MSn spectrum provides more structurally specific information as the MSn stage advances, therefore, resulting in a confident and detailed structural identification. The structures of minute ion species that are often difficult to define by other analytical method can also be assigned (Table 1). However, the sensitivity declines as the higher order of MSn stage proceeds. Other drawback includes that a complete structural information is not necessary extractable by MSn. For example, the positions of the double bond and methyl side chain of the fatty acid substituents at C3 have not been defined in this study.

The LIT MSn approach as described here affords near complete structural characterization of a complex DAT lipid family, locating the fatty acyl groups on the trehalose backbone, and recognizing many isomeric structures. A LIT MSn approach combined with chemical reaction modification [9,34] for locating the functional groups including the methyl, hydroxyl, and the double bond on the fatty acid substituents are currently in progress in our laboratory.

**Author Contributions:** C.F. performs lipid separation; R.B.A. and G.E.P. grow cell and extract total lipids; L.L. and L.L. synthesize DAT standards. F.-F.H. performs mass spectrometry analysis and write the manuscript.

**Funding:** This research was funded by US Public Health Service Grants P41GM103422, P30DK020579, R01AI130454, R21AI128427, R21HL120760, and R21AI113074.

**Acknowledgments:** This research was supported by US Public Health Service Grants P41GM103422, P30DK020579, R01AI130454, R21AI128427, R21AI113074 and R21HL120760.

**Conflicts of Interest:** The authors declare no conflict of interest.
