May 09, 2024

Public workspaceUntargeted lipidomics of Tagless Lyso-IP

The Journal of Clinical Investigation
DOI
dx.doi.org/10.17504/protocols.io.yxmvm3r8bl3p/v1
  • Wentao Dong1,2,
  • Eshaan S Rawat1,2,
  • Daniel Saarela3,2,
  • Monther Abu-Remaileh1,2
  • 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
Francesca Tonelli
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DOI: dx.doi.org/10.17504/protocols.io.yxmvm3r8bl3p/v1
Protocol Citation: Wentao Dong, Eshaan S Rawat, Daniel Saarela, Monther Abu-Remaileh 2024. Untargeted lipidomics of Tagless Lyso-IP. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3r8bl3p/v1
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 Acknowledgements:
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.
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Materials
Reagents

  • ReagentWater, Optima™ LC/MS Grade, Fisher Chemical™Thermo Fisher ScientificCatalog #AAB-W6-4
  • ReagentAcetonitrile, Optima™ LC/MS Grade, Fisher Chemical™Thermo Fisher ScientificCatalog #AAB-A955-4
  • ReagentIsopropanol, Optima™LC/MS Grade, Fisher Chemical™Thermo Fisher ScientificCatalog #AAB-A461-500
  • Ammonium formate
  • Formic acid
  • EASYICTM
  • Splashmix (SPLASH® LIPIDOMIX® Mass Spec Standard, cat. no. 330707)
  • ReagentSodium chloride 0.9% in aqueous solution Normal saline solution, sterileAvantor SciencesCatalog #S5825 (101320-574)

Equipment

  • ID-X Orbitrap Tribrid Mass Spectrometer (Thermo Fisher Scientific) with a heated electrospray
  • ionization (HESI) probe
Equipment
Ascentis® Express C18, 2.7 μm HPLC Column
NAME
HPLC Column
TYPE
Ascentis®
BRAND
53825-U
SKU
https://www.sigmaaldrich.com/IN/en/product/supelco/53825u
LINK

Equipment
Ascentis® Express Guard Cartridge Holder
NAME
for use with Ascentis Express Guard Columns, pk of 1
TYPE
Ascentis®
BRAND
53500-U
SKU
https://www.sigmaaldrich.com/IN/en/product/supelco/53500u
LINK

Equipment
DynaMag™- Spin Magnet
NAME
Invitrogen™
BRAND
12320D
SKU
https://www.thermofisher.com/order/catalog/product/12320D
LINK

  • Microcentrifuge with thermostat (VWR Micro Star 17R. S/N 42209232. REF# 521-1647)
  • Eppendorf ThermoMixer‱ C, Eppendorf, #EP02095
Equipment
ThermoTop®
NAME
ThermoTop®
BRAND
5308000003
SKU
https://www.eppendorf.com/us-en/eShop-Products/Temperature-Control-and-Mixing/Accessories/ThermoTop-p-5308000003
LINK

Equipment
Savant™ SpeedVac™ Medium Capacity Vacuum Concentrators for Combinatorial Chemistry Applications
NAME
Thermo Scientific™
BRAND
SPD140DDA-115
SKU
https://www.thermofisher.com/order/catalog/product/SPD140DDA-115
LINK
C18-based lipid separation

Buffer A
AB
Ammonium formate10 millimolar (mM)
Formic acid 0.1 % (v/v)
Dissolve in
LC/MS grade waterDissolve in 60 % (v/v)
LC/MS grade acetonitrile Dissolve in 40 % (v/v)

Buffer B
AB
Ammonium formate10 millimolar (mM)
Formic acid 0.1 % (v/v)
Dissolve in
LC/MS grade 2-propanol 90 % (v/v)
LC/MS grade acetonitrile10 % (v/v)




Untargeted lipidomics of Tagless Lyso-IP
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/v1 (Tagless Lyso-IP).

Note
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 Amount1000 µL of chloroform:methanol at ratio of 2:1 (v/v) with 1000x diluted Splashmix (Avanti).

Pipetting
Incubate at Temperature4 °C for Duration00:10:00 .

10m
Incubation
Place the Tagless Lyso-IP samples on a tube magnet for Duration00:00:30 and transfer the supernatant to a fresh 1.5 mL Eppendorf tube.

30s
Pipetting
Vortex both the Tagless Lyso-IP samples and their corresponding whole cell samples (from Step 1) at Temperature4 °C for Duration01:00:00 .

1h
Add Amount200 µL of 0.9% (w/v) saline (VWR) and vortex at Temperature4 °C for Duration00:10:00 .

10m
Pipetting
Centrifuge all samples at Centrifigation3000 x g , Temperature4 °C for Duration00:05:00 .

5m
Centrifigation
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 Temperature-80 °C .

On the day of analysis reconstitute the dried lipid extracts in Amount50 µL of ACN:IPA:water 13:6:1 (v/v/v).

Pipetting
Vortex at Temperature4 °C for Duration00:10:00 .

10m
Centrifuge at Centrifigation13000 x g , Temperature4 °C for Duration00:15:00 .

15m
Centrifigation
Insert Amount45 µL of supernatant into glass insert vials for LC/MS.

Pipetting
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 Temperature300 °C , and vaporizer temperature to Temperature375 °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

AB
Cycle time1.5 sec
Microscans1 unit
Isolation windowm/z 1
Intensity threshold1 x 104
Dynamic exclusion time2.5 sec

For HCD fragmentation, use step-wise collision energies of 15%, 25%, and 35%.
Perform the elution with a gradient of Duration00:40:00 :

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.

AB
Mass tolerance10 ppm
Minimum and maximum precursor mass0-5,000 Da
Retention time limit0.1-30 min
Peak filter signal to noise ratio1.5
Retention time alignment maximum shift1 min
Minimum peak intensity10,000
Compound detection signal to noise ratio3
Isotope and adduct settingsDefault 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.