Untargeted metabolomics of Tagless Lyso-IP

This method is following successful isolation of lysosomes the Tagless Lyso-IP method as described

in:dx.doi.org/10.17504/protocols.io.x54v9yp51g3e/v11 (Tagless Lyso-IP).

Resuspend the lysosomes attached to the magnetic beads and the pelleted whole cell samples in 50µL of 80% MeOH (v/v) with isotopically labelled amino acids and vortex briefly.

Resuspend your whole cell pellets in 225µL of 80% MeOH (v/v) with isotopically labelled amino acids and vortex briefly.

Incubate at 4°C for 0h 10m 0s.

Place your Lyso-IP samples (Step 1) on a tube magnet for 0h 0m 30s.

Transfer the supernatant into a fresh 1.5 mL Eppendorf tube.

Centrifuge both the Lyso-IP and the whole cell samples (Steps 2 and 5) at 13000x g,0h 0m 0s, 4°C

for0h 10m 0s .

Transfer the supernatant to fresh 1.5 mL Eppendorf tubes and store at -80°C.

On the day of LC/MS measurement, vortex samples at 4°C for 0h 10m 0s.

Centrifuge the samples at 13000x g,0h 0m 0s, 4°C for 0h 10m 0s.

Transfer the supernatant to autosampler vials.

Set an ID-X tribrid mass spectrometer (Thermo Fisher Scientific) with an electrospray ionization (ESI) probe, for initial polar metabolite profiling.

Prepare a SeQuant® ZIC®-pHILIC 150 x 2.1 mm column (Millipore Sigma 1504600001) coupled with a 20 x 2.1 mm (Millipore Sigma 1504380001) guard, to carry out hydrophilic interaction chromatography (HILIC) for metabolite separation prior to mass spectrometry. Use EASYIC™ for internal calibration.

For HILIC metabolite separation, use 20 millimolar (mM) ammonium carbonate and 0.1 % (v/v) ammonium hydroxide dissolved in 100 % (v/v) LC/MS grade water for Buffer A, and 100 % (v/v) LC/MS grade acetonitrile for Buffer B..

Run the chromatographic gradient at a flow rate of 0.150 mL/min. Operate the mass spectrometer in full-scan, polarity-switching mode at m/z 70-1000, with Orbitrap resolution set at 120,000, RF lens at 40%, AGC target at 1x106, and maximum injection time at 80 ms. Set positive ion voltage to 3000 V, negative ion voltage to 2500 V, ion transfer tube temperature to 275°C, and vaporizer temperature to 350°C. Set sheath gas flow to 40 units, auxiliary gas flow to 15 units, and sweep gas flow to 1 unit.

For unbiased differential analysis, extract ion chromatograms using Compound Discoverer (Thermo Fisher Scientific) with a mass tolerance of 5 ppm. Rigorously quantify metabolite abundance using TraceFinder (Thermo Fisher Scientific) in conjunction with an in-house library of known metabolite standards (MSMLS, Sigma-Aldrich).

Compound Discoverer (Thermo Fisher Scientific) was used for initial unbiased differential analysis. In addition to online databases,we also included a local library with both masslist and mzVault spectral archives. Mass tolerance for untargeted discovery, 10 ppm; minimum and maximum precursor mass, 0-5,000 Da; retention time limit, 0-20 min; Peak filter signal to noise ratio, 1.5; retention time alignment maximum shift, 0.5 min; minimum peak intensity, 10,000; compound detection signal to noise ratio, 3. Isotope and adduct settings were kept at default values. Gap filling and background filtering were performed by default settings. Area normalization was performed by constant median after blank exclusion. Compound annotation priority: #1, MassList Search; #2, mzVault Search; #3, mzCloud Search; #4, Predicted Compositions; #5, Chemspider Search; #6, Metabolika Search.

The MassList Search was customized with 5 ppm mass tolerance and 1 minute retention time tolerance. * The mzVault Search was customized with 10 ppm precursor and fragment mass tolerance and 1 minute retention time tolerance.

• The mzCloud Search was customized with 10 ppm precursor and fragment mass tolerance.
• The other searches were performed with default parameters specified in the default workflow “Untargeted Metabolomics with Statistics Detect Unknowns with ID using Online Databases and mzLogic” provided by Compound Discoverer.

Raw features were further filtered by the following algorithm:

1. MS2 fragmentation spectra were obtained.
2. At least 1 annotation match in the mzVault, mzCloud or Chemspider Search.

To further improve the rigor of our discovery workflow, we performed additional manual filtering based on the following criteria:

• Features with retention time earlier than 3 minutes on this HILIC column, which are nonpolar and should be quantified by a C18 column, were removed.
• Features with predicted compositions containing chemical elements rarely found in human metabolome (e.g. certain halogens) were removed.
• Features enriched in the lysosomes from only one independent experiment were removed.

Rigorous quantification of metabolite abundance was performed by TraceFinder (Thermo Fisher Scientific) in conjunction with an in-house library of known metabolite standards (MSMLS, Sigma-Aldrich). Isotopically labelled amino acids were used as internal standards. Mass tolerance for extracting ion chromatograms, 5 ppm.