Lipidome

The phagosomal lipid extractions were performed using an established
protocol.^{16 (link),17 (link),28 (link)} Briefly, the
phagosomal preparations were washed with sterile Dulbecco’s
PBS (DPBS; three times) and transferred into a glass vial using 1
mL of DPBS. A total of 3 mL of 2:1 (v/v) chloroform (CHCl₃)/methanol (MeOH) with the internal standard mix (50 pmol of each
internal standard listed in Supporting Information Table 1) was added, and the mixture was vortexed. The two phases
were separated by centrifugation at 2800g for 5 min.
The organic phase (bottom) was removed. A total of 50 μL of
formic acid was added to acidify the aqueous homogenate, and CHCl₃ was added to make up a 4 mL volume. The mixture was vortexed,
and separated by centrifugation at 2800g for 5 min.
Both the organic extracts were pooled and dried under a stream of
N₂. The lipidome was solubilized in 200 μL of 2:1
(v/v) CHCl₃/MeOH, and 20 μL was used for the lipidomics
analysis. All the lipid species analyzed in this study were quantified
using the multiple reaction monitoring high resolution (MRM-HR) scanning
method (Supporting Information Table 1)
on a Sciex X500R QTOF mass spectrometer (MS) fitted with an Exion-LC
series UHPLC. All data were acquired and analyzed using the SciexOS
software. The LC separation was achieved using a Gemini 5U C18 column
(Phenomenex, 5 μm, 50 × 4.6 mm) coupled to a Gemini guard
column (Phenomenex, 4 × 3 mm). The LC solvents were as follows:
positive mode, buffer A, 95:5 (v/v) H₂O/MeOH + 0.1% formic
acid + 10 mM ammonium formate; buffer B, 60:35:5 (v/v) isopropanol
(IPA)/MeOH/H₂O + 0.1% (v/v) formic acid + 10 mM ammonium
formate; negative mode, buffer A, 95:5 (v/v) H₂O/MeOH +
0.1% (v/v) NH₄OH; buffer B, 60:35:5 (v/v) IPA/MeOH/H₂O + 0.1% (v/v) NH₄OH. All the lipid estimations
were performed using an electrospray ion (ESI) source, with following
MS parameters: turbo spray ion source, medium collision gas, curtain
gas = 20 L min^–1, ion spray voltage = 4500 V (positive
mode) or −5500 V (negative mode), at 400 °C. A typical
LC-run was 55 min, with the following solvent run sequence post injection:
0.3 mL min^–1 0% B for 5 min, 0.5 mL min^–1 0% B for 5 min, 0.5 mL min^–1 linear gradient of
B from 0–100% over 25 min, 0.5 mL min^–1 of
100% B for 10 min, and re-equilibration with 0.5 mL min^–1 of 0% B for 10 min. A detailed list of all the species targeted
in this MRM-HR study, describing the precursor ion mass and adduct,
the product ion targeted, and MS voltage parameters can be found in Supporting Information Table 1. All the endogenous
lipid species were quantified by measuring the area under the curve
in comparison to the respective internal standard, and then normalizing
to the total protein content of the phagosomal preparation. All the
lipidomics data are represented as mean ± SEM of four (or more)
biological replicates per group (Supporting Information Table 1).