*2.1. Materials*

All solvents (spectroscopic grade) and chemicals (ACS grade) were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

## *2.2. Sample Preparation*

*M. tuberculosis* strain H37Rv were grown and total lipids were extracted and isolated as previously described [8]. Briefly, the total lipid was separated by a Phenomenex C18 Kinetex (100 × 4.6 mm, pore size 100 Å, particle size 2.6 μm) column at a flow rate of 300 μL/min with a gradient system as previously described [12]. DAT (eluted at 24.45–26.43 min) fraction from three injections (~200 μg total lipid/injection) were collected and pooled, dried under a stream of nitrogen, and stored at −20 ◦C until use.

## *2.3. Mass Spectrometry*

Both high-resolution (R = 100,000 at m/z 400) low-energy CID and higher collision-energy dissociation (HCD) tandem mass spectrometric experiments were conducted on a Thermo Scientific (San Jose, CA, USA) LTQ Orbitrap Velos mass spectrometer (MS) with Xcalibur operating system. Samples in CH3OH were infused (1.5 μL/min; ~10 pmol/μL) to the ESI source, where the skimmer was set at ground potential, the electrospray needle was set at 4.0 kV, and temperature of the heated capillary was 300 ◦C. The automatic gain control of the ion trap was set to 5 × 104, with a maximum injection time of 100 ms. Helium was used as the buffer and collision gas at a pressure of 1 × 10−<sup>3</sup> mbar (0.75 mTorr). The MSn experiments were carried out with an optimized relative collision energy ranging from 25–35% and with an activation q value at 0.25. The activation time was set for 10 ms to leave a minimal residual abundance of precursor ion (around 20%). For the HCD experiments, the collision energy was set at 50–55% and mass scanned from m/z 100 to the upper m/z value that covers the precursor ions. The mass selection window for the precursor ions was set at 1 Da wide to admit the monoisotopic peak to the ion-trap for collision-induced dissociation (CID) for unit resolution detection in the ion-trap or high resolution accurate mass detection in the Orbitrap mass analyzer. Mass spectra were accumulated in the profile mode, typically for 3–10 min for MSn spectra (n = 2,3,4).
