Untargeted lipidomics of Tagless Lyso-IP
Wentao Dong, Eshaan S Rawat, Monther Abu-Remaileh, Daniel Saarela
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Abstract
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.
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Untargeted lipidomics 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).
Processing of nonpolar metabolite samples (lipids)
Resuspend the lysosomes attached to the magnetic beads and the pelleted whole cell samples in 1000µL of chloroform:methanol at ratio of 2:1 (v/v) with 1000x diluted Splashmix (Avanti).
Incubate at 4°C for 0h 10m 0s.
Place the Tagless Lyso-IP samples on a tube magnet for 0h 0m 30s and transfer the supernatant to a fresh 1.5 mL Eppendorf tube.
Vortex both the Tagless Lyso-IP samples and their corresponding whole cell samples (from Step 1) at 4°C for 1h 0m 0s.
Add 200µL of 0.9% (w/v) saline (VWR) and vortex at 4°C for 0h 10m 0s.
Centrifuge all samples at 3000x g,0h 0m 0s, 4°C for 0h 5m 0s.
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.
Vacuum dry the samples and store at -80°C.
On the day of analysis reconstitute the dried lipid extracts in 50µL of ACN:IPA:water 13:6:1 (v/v/v).
Vortex at 4°C for 0h 10m 0s.
Centrifuge at 13000x g,0h 0m 0s, 4°C for 0h 15m 0s.
Insert 45µL of supernatant into glass insert vials for LC/MS.
LC/MS lipidomics settings
Set an ID-X tribrid mass spectrometer (Thermo Fisher Scientific) with a heated electrospray ionization (HESI) probe, for initial nonpolar lipid profiling.
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.
For C18-based lipid separation, for buffer preparation refer to the material section.
Set the chromatographic gradient flow rate to 0.26 mL/min.
Use Orbitrap resolution 120,000 for MS1 and 30,000 for MS2.
Use RF lens at 40%.
Use AGC target 4x105 for MS1 and 5x104 for MS2.
Use maximum injection time 50 ms for MS1 and 54 ms for MS2.
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.
Set sheath gas flow to 40 units, auxiliary gas flow to 10 units, and sweep gas flow to 1 unit.
Operate the mass spectrometer in full-scan mode with data-dependent tandem mass spectrometry (ddMS2) at m/z 250-1500, with
| A | B |
|---|---|
| Cycle time | 1.5 sec |
| Microscans | 1 unit |
| Isolation window | m/z 1 |
| Intensity threshold | 1 x 104 |
| Dynamic exclusion time | 2.5 sec |
For HCD fragmentation, use step-wise collision energies of 15%, 25%, and 35%.
Perform the elution with a gradient of 0h 40m 0s:
From 0−1.5 min isocratically elute at 32% B.
From 35-35.1 min linearly decrease to 32% B.
From 35.1-40 min hold at 32% min.
From 1.5-4 min linearly increase to 45% B.
From 4-5 min linearly increase to 52% B.
From 5-8 min linearly increase to 58% B.
From 8-11 min linearly increase to 66% B.
From 11-14 min linearly increase to 70%.
From 14-18 min linearly increase to 75%.
From 18- 21 min linearly increase to 97% B.
From 21-35 min hold at 97% B.
Untargeted lipidomics workflow
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.
The mass list from LipidSearch is then exported and used in Compound Discoverer for improved alignment and quantitation.
| A | B |
|---|---|
| Mass tolerance | 10 ppm |
| Minimum and maximum precursor mass | 0-5,000 Da |
| Retention time limit | 0.1-30 min |
| Peak filter signal to noise ratio | 1.5 |
| Retention time alignment maximum shift | 1 min |
| Minimum peak intensity | 10,000 |
| Compound detection signal to noise ratio | 3 |
| Isotope and adduct settings | Default values |
| Gap filling and background filtering | Default settings |
