1Department of Chemical Engineering, Stanford University, Stanford, CA 94305.;
2Aligning Science Across Parkinson's Collaborative Research Network, Chevy Chase, MD 20815.;
3Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 20, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95584
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
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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.
In the steps following the immunoprecipitation of lysosomes (Steps 28-32) the wash buffer used is ice-cold KPBS without protease and phosphatase inhibitors.
Processing of nonpolar metabolite samples (lipids)
Processing of nonpolar metabolite samples (lipids)
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).
Incubate at 4 °C for 00:10:00.
10m
Place the Tagless Lyso-IP samples on a tube magnet for 00:00:30 and transfer the supernatant to a fresh 1.5 mL Eppendorf tube.
30s
Vortex both the Tagless Lyso-IP samples and their corresponding whole cell samples (from Step 1) at 4 °C for 01:00:00.
1h
Add 200 µL of 0.9% (w/v) saline (VWR) and vortex at 4 °C for 00:10:00.
10m
Centrifuge all samples at 3000 x g, 4 °C for 00:05:00.
5m
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 50 µL of ACN:IPA:water 13:6:1 (v/v/v).
Vortex at 4 °C for 00:10:00.
10m
Centrifuge at 13000 x g, 4 °C for 00:15:00.
15m
Insert 45 µL of supernatant into glass insert vials for LC/MS.
LC/MS lipidomics settings
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 00:40:00:
40m
From 0−1.5 min isocratically elute at 32% B.
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.
From 35-35.1 min linearly decrease to 32% B.
From 35.1-40 min hold at 32% min.
Untargeted lipidomics workflow
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
Note
The MassList Search was customized with 5 ppm mass tolerance and 1 minute retention time tolerance.
Area normalization was performed by constant median after blank exclusion.