Dec 16, 2024

Public workspaceLipidomic Analysis of Neuronal Lipid Droplets

DOI
dx.doi.org/10.17504/protocols.io.14egn99kpl5d/v1
  • Mukesh Kumar1,
  • Justin Knapp2,
  • Kallol Gupta3,2,4,
  • Timothy A. Ryan1,3
  • 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065;
  • 2Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA;
  • 4Nanobiology Institute, Yale University, West Haven, CT 06516, USA
Mukesh Kumar
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DOI: dx.doi.org/10.17504/protocols.io.14egn99kpl5d/v1
Protocol Citation: Mukesh Kumar, Justin Knapp, Kallol Gupta, Timothy A. Ryan 2024. Lipidomic Analysis of Neuronal Lipid Droplets. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn99kpl5d/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: November 27, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 115703
Keywords: ASAPCRN
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol provides details of extraction, identification, and quantification of lipids from purified lipid droplets using advanced LC-MS techniques, enabling detailed analysis of constituent lipids.
Guidelines
The protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
  • LD samples: Collected as per LD purification protocol
  • MTBE (Methyl tert-butyl ether)
  • Avanti Splashmix (Internal standards)
  • 1-Butanol
  • Mobile Phase A: 60% Acetonitrile, 40% H₂O, 7.5 mM Ammonium Acetate
  • Mobile Phase B: 90% Isopropanol (IPA), 10% Acetonitrile, 7.5 mM Ammonium Acetate
  • MS-DIAL software (version 4.7)

Equipments

• Centrifuge capable of processing organic phase extractions
• LC-MS system with gradient capability
• Agilent Poroshell 120 EC-C18 column (2.7 μm, 1000 bar, 2.1 × 100 mm)
• Agilent 1290 Infinity II LC system
• Agilent Quadrupole Time-Of-Flight 6546 mass spectrometer
Lipid Extraction
Lipid Extraction
Prepare the LD samples for extraction by ensuring they are thawed TemperatureOn ice and homogenized.

Temperature
Add MTBE to the LD samples along with spiked Avanti Splashmix (internal standards) to normalize lipid quantification.
Vortex the mixture thoroughly to ensure complete mixing.
Mix
Centrifuge the mixture to separate the organic phase.
Centrifigation
Collect the organic phase carefully to avoid contamination from the aqueous phase.
Dry the collected organic phase under a gentle stream of nitrogen or in a vacuum concentrator.
Lipid Resuspension
Lipid Resuspension
Resuspend the dried lipid extract in 1-Butanol.
Vortex the sample to ensure uniform suspension.
Mix
Load Amount3 µL of the lipid mixture onto the LC-MS system.

Pipetting
LC-MS Analysis
LC-MS Analysis
17m 30s
17m 30s
Set up the Agilent Poroshell 120 EC-C18 column (2.7 μm, 2.1 × 100 mm) on the Agilent 1290 Infinity II LC system.
Prepare the following mobile phases:

Mobile Phase A (60% Acetonitrile, 40% H₂O, 7.5 mM Ammonium Acetate),
Mobile Phase B (90% IPA, 10% Acetonitrile, 7.5 mM Ammonium Acetate).
Use the following gradient for lipid separation:

  1. Start at 85% A (15% B) and decrease to 70% A over Duration00:02:00 .
  2. Further decrease to 52% A over Duration00:00:30 .
  3. Gradually reduce to 18% A over Duration00:05:00 .
  4. Reduce to 1% A within Duration00:01:00 and hold for Duration00:04:00 .

12m 30s
Restore gradient to 85% A and wash the column for Duration00:05:00 .

5m
Wash
Perform the analysis on the Agilent Quadrupole Time-Of-Flight 6546 mass spectrometer for high-resolution detection of lipid species.
Data Analysis
Data Analysis
Process the LC-MS data using MS-DIAL software (version 4.7; PMID: 25938372).
Identify lipid species based on the mass spectra and retention times.
Quantify each lipid species using the moles of corresponding class-specific lipid standards present in the Splashmix.

Note
• Ensure the organic phase is free from aqueous contamination to avoid interference in LC-MS.
• Handle MTBE and other organic solvents with care in a fume hood to prevent exposure.
• The gradient setup must be precise to ensure reproducible lipid separation and identification.