TITLE: Untargeted lipidomics of Tagless Lyso-IP
AUTHORS: Wentao Dong, Eshaan S Rawat, Daniel Saarela, Monther Abu-Remaileh

[DESCRIPTION]
Lysosomal biology is increasingly implicated in neurodegenerative diseases and health. It has traditionally been difficult to profile the metabolomic homeostasis of the lysosome in disease states. To overcome this challenge we have developed the Tagless Lyso-IP method to rapidly prepare lysosome enriched samples from human peripheral blood. This protocol details the processing and untargeted analysis of nonpolar metabolites derived using the Tagless Lyso-IP method. 

[STEPS]
SECTION: Untargeted lipidomics of Tagless Lyso-IP
1. This method is following successful isolation of lysosomes the Tagless Lyso-IP method as described
in: dx.doi.org/10.17504/protocols.io.x54v9yp51g3e/v1 (Tagless Lyso-IP). 

 

SECTION: Processing of nonpolar metabolite samples (lipids)
2. Resuspend the lysosomes attached to the magnetic beads and the pelleted whole cell samples in 1000 µL of chloroform:methanol at ratio of 2:1 (v/v) with 1000x diluted Splashmix (Avanti). 

SECTION: Processing of nonpolar metabolite samples (lipids)
2.1. Incubate at 4 °C for 10 min.

SECTION: Processing of nonpolar metabolite samples (lipids)
3. Place the Tagless Lyso-IP samples on a tube magnet for 30 s and transfer the supernatant to a fresh 1.5 mL Eppendorf tube.

SECTION: Processing of nonpolar metabolite samples (lipids)
4. Vortex both the Tagless Lyso-IP samples and their corresponding whole cell samples (from Step 1) at 4 °C for 60 min.

SECTION: Processing of nonpolar metabolite samples (lipids)
5. Add 200 µL of 0.9% (w/v) saline (VWR) and vortex at 4 °C for 10 min.

SECTION: Processing of nonpolar metabolite samples (lipids)
6. Centrifuge all samples at 3000 x g, 4 °C for 5 min.

SECTION: Processing of nonpolar metabolite samples (lipids)
7. Discard the top layer (MeOH and saline polar phase) and use the bubbling method to retrieve the bottom layer of chloroform containing the lipids to a fresh 1.5 mL Eppendorf tube.
SECTION: Processing of nonpolar metabolite samples (lipids)
8. Vacuum dry the samples and store at -80 °C.

SECTION: Processing of nonpolar metabolite samples (lipids)
9. On the day of analysis reconstitute the dried lipid extracts in 50 µL of ACN:IPA:water 13:6:1 (v/v/v).

SECTION: Processing of nonpolar metabolite samples (lipids)
10. Vortex at 4 °C for 10 min.

SECTION: Processing of nonpolar metabolite samples (lipids)
11. Centrifuge at 13000 x g, 4 °C for 15 min.

SECTION: Processing of nonpolar metabolite samples (lipids)
12. Insert 45 µL of supernatant into glass insert vials for LC/MS.

SECTION: LC/MS lipidomics settings
13. Set an ID-X tribrid mass spectrometer (Thermo Fisher Scientific) with a heated electrospray ionization (HESI) probe, for initial nonpolar lipid profiling.
SECTION: LC/MS lipidomics settings
14. Prepare an Ascentis Express C18 150 x 2.1 mm column (Millipore Sigma 53825-U) coupled with a 5 x 2.1 mm guard (Sigma-Aldrich 53500-U), to carry out C18-based lipid separation prior to mass spectrometry. Use EASYICTM for internal calibration.
SECTION: LC/MS lipidomics settings
15. For C18-based lipid separation, for buffer preparation refer to the material section.
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16. Set the chromatographic gradient flow rate to 0.26 mL/min.
SECTION: LC/MS lipidomics settings
17. Operate the mass spectrometer in full-scan mode with data-dependent tandem mass spectrometry (ddMS2) at m/z 250-1500, with

 

SECTION: LC/MS lipidomics settings
18. Perform the elution with a gradient of 40 min:

SECTION: Untargeted lipidomics workflow
19. LipidSearch and Compound Discoverer (Thermo Fisher Scientific) were used for unbiased
differential analysis. Lipid annotation was acquired from LipidSearch with the precursor
tolerance at 5 ppm and product tolerance at 8 ppm.
SECTION: Untargeted lipidomics workflow
20. The mass list from LipidSearch is then exported and used in Compound Discoverer for improved alignment and quantitation. 

 

 

SECTION: LC/MS lipidomics settings
16.1. Use Orbitrap resolution 120,000 for MS1 and 30,000 for MS2.
SECTION: LC/MS lipidomics settings
16.5. Set positive ion voltage to 3250 V, negative ion voltage to 3000 V, ion transfer tube temperature to 300 °C, and vaporizer temperature to 375 °C.

SECTION: LC/MS lipidomics settings
16.6. Set sheath gas flow to 40 units, auxiliary gas flow to 10 units, and sweep gas flow to 1 unit. 
SECTION: LC/MS lipidomics settings
18.1. From 0−1.5 min isocratically elute at 32% B.
SECTION: LC/MS lipidomics settings
18.2. From 1.5-4 min linearly increase to 45% B.
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18.3. From 4-5 min linearly increase to 52% B.
SECTION: LC/MS lipidomics settings
18.5. From 8-11 min linearly increase to 66% B.
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18.4. From 5-8 min linearly increase to 58% B.
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18.6. From 11-14 min linearly increase to 70%.
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18.7. From 14-18 min linearly increase to 75%.
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18.8. From 18- 21 min linearly increase to 97% B.
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18.9. From 21-35 min hold at 97% B.
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18.10. From 35-35.1 min linearly decrease to 32% B.
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18.11. From 35.1-40 min hold at 32% min.
SECTION: LC/MS lipidomics settings
17.1. For HCD fragmentation, use step-wise collision energies of 15%, 25%, and 35%.
SECTION: LC/MS lipidomics settings
16.3. Use AGC target 4x105 for MS1 and 5x104 for MS2.
SECTION: LC/MS lipidomics settings
16.4. Use maximum injection time 50 ms for MS1 and 54 ms for MS2.
SECTION: LC/MS lipidomics settings
16.2. Use RF lens at 40%.