May 09, 2024

Public workspaceUntargeted metabolomics of Tagless Lyso-IP

DOI
dx.doi.org/10.17504/protocols.io.kxygx3jm4g8j/v1
  • Wentao Dong1,2,
  • Eshaan S Rawat1,2,
  • Daniel Saarela1,2,
  • Monther Abu-Remaileh1,2
  • 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom;
  • 2Aligning Science Across Parkinson's Collaborative Research Network, Chevy Chase, MD 20815.
Francesca Tonelli
Open access
DOI: dx.doi.org/10.17504/protocols.io.kxygx3jm4g8j/v1
Protocol Citation: Wentao Dong, Eshaan S Rawat, Daniel Saarela, Monther Abu-Remaileh 2024. Untargeted metabolomics of Tagless Lyso-IP. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3jm4g8j/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: 95656
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 polar metabolites derived using the Tagless Lyso-IP method.
Attachments
1024-2637.pdf
1024-2637.pdf
89KB
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
  • Ammonium carbonate
  • Ammonium hydroxide
  • EASYICTM
  • ReagentMetabolomics Amino Acid MixCambridge Isotope Laboratories, Inc.Catalog #MSK-A2-1.2
Equipment

  • ID-X Orbitrap Tribrid Mass Spectrometer (Thermo Fisher Scientific) with an electrospray ionization (ESI) probe

Equipment
SeQuant® ZIC®-pHILIC 5µm polymer 150 x 2.1 mm
NAME
SeQuant®
BRAND
1.50460
SKU
https://www.sigmaaldrich.com/IN/en/product/mm/150460
LINK

Equipment
SeQuant® ZIC®-pHILIC Guard Kit 20 x 2.1 mm
NAME
SeQuant®
BRAND
150438
SKU
https://www.merckmillipore.com/IN/en/product/SeQuant-ZIC-pHILIC-Guard-Kit-20-x-2.1-mm,MDA_CHEM-150438
LINK

Equipment
Dynamag-50 Separation Magnet
NAME
Magnet
TYPE
Thermo Fisher Scientific
BRAND
12302D
SKU

  • Microcentrifuge with thermostat (VWR Micro Star 17R. S/N 42209232. REF# 521-1647)
  • Eppendorf ThermoMixer‱ C, Eppendorf, #EP02095

Equipment
ThermoTop®
NAME
Smart block
TYPE
Eppendorf
BRAND
5308000003
SKU
HILIC metabolite separation
Buffer A
AB
Ammonium carbonate 20 millimolar (mM)
Ammonium hydroxide0.1 % (v/v)
Dissolve in
LC/MS grade water100 % (v/v)

Buffer B
AB
Ammonium carbonate 20 millimolar (mM)
Ammonium hydroxide0.1 % (v/v)
Dissolve in
LC/MS grade acetonitrile100 % (v/v)



Untargeted metabolomics of Tagless Lyso-IP
Untargeted metabolomics 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
The for 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 polar metabolite samples
Processing of polar metabolite samples
Resuspend the lysosomes attached to the magnetic beads and the pelleted whole cell samples in Amount50 µL of 80% MeOH (v/v) with isotopically labelled amino acids and vortex briefly.

Pipetting
Resuspend your whole cell pellets in Amount225 µL of 80% MeOH (v/v) with isotopically labelled amino acids and vortex briefly.

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

10m
Incubation
Place your Lyso-IP samples (Step 1) on a tube magnet for Duration00:00:30 .

30s
Transfer the supernatant into a fresh 1.5 mL Eppendorf tube.
Pipetting
Centrifuge both the Lyso-IP and the whole cell samples (Steps 2 and 5) at Centrifigation13000 x g , Temperature4 °C for Duration00:10:00 .

10m
Centrifigation
Transfer the supernatant to fresh 1.5 mL Eppendorf tubes and store at Temperature-80 °C .

Pipetting
On the day of LC/MS measurement, vortex samples at Temperature4 °C for Duration00:10:00 .

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

10m
Centrifigation
Transfer the supernatant to autosampler vials.
Pipetting
LC/MS metabolomics settings
LC/MS metabolomics settings
Set an ID-X tribrid mass spectrometer (Thermo Fisher Scientific) with an electrospray ionization (ESI) probe, for initial polar metabolite profiling.
Prepare a SeQuant® ZIC®-pHILIC 150 x 2.1 mm column (Millipore Sigma 1504600001) coupled with a 20 x 2.1 mm (Millipore Sigma 1504380001) guard, to carry out hydrophilic interaction chromatography (HILIC) for metabolite separation prior to mass spectrometry. Use EASYIC™ for internal calibration.
For HILIC metabolite separation, use 20 millimolar (mM) ammonium carbonate and 0.1 % (v/v) ammonium hydroxide dissolved in 100 % (v/v) LC/MS grade water for Buffer A, and 100 % (v/v) LC/MS grade acetonitrile for Buffer B..
Run the chromatographic gradient at a flow rate of 0.150 mL/min. Operate the mass spectrometer in full-scan, polarity-switching mode at m/z 70-1000, with Orbitrap resolution set at 120,000, RF lens at 40%, AGC target at 1x106, and maximum injection time at 80 ms. Set positive ion voltage to 3000 V, negative ion voltage to 2500 V, ion transfer tube temperature to Temperature275 °C , and vaporizer temperature to Temperature350 °C . Set sheath gas flow to 40 units, auxiliary gas flow to 15 units, and sweep gas flow to 1 unit.

For unbiased differential analysis, extract ion chromatograms using Compound Discoverer (Thermo Fisher Scientific) with a mass tolerance of 5 ppm. Rigorously quantify metabolite abundance using TraceFinder (Thermo Fisher Scientific) in conjunction with an in-house library of known metabolite standards (MSMLS, Sigma-Aldrich).
Compound Discoverer (Thermo Fisher Scientific) was used for initial unbiased differential analysis. In addition to online databases,we also included a local library with both masslist and mzVault spectral archives. Mass tolerance for untargeted discovery, 10 ppm; minimum and maximum precursor mass, 0-5,000 Da; retention time limit, 0-20 min; Peak filter signal to noise ratio, 1.5; retention time alignment maximum shift, 0.5 min; minimum peak intensity, 10,000; compound detection signal to noise ratio, 3. Isotope and adduct settings were kept at default values. Gap filling and background filtering were performed by default settings. Area normalization was performed by constant median after blank exclusion. Compound annotation priority: #1, MassList Search; #2, mzVault Search; #3, mzCloud Search; #4, Predicted Compositions; #5, Chemspider Search; #6, Metabolika Search.
  • The MassList Search was customized with 5 ppm mass tolerance and 1 minute retention time tolerance.
  • The mzVault Search was customized with 10 ppm precursor and fragment mass tolerance and 1 minute retention time tolerance.
  • The mzCloud Search was customized with 10 ppm precursor and fragment mass tolerance.
  • The other searches were performed with default parameters specified in the default workflow “Untargeted Metabolomics with Statistics Detect Unknowns with ID using Online Databases and mzLogic” provided by Compound Discoverer.
Raw features were further filtered by the following algorithm:

  1. MS2 fragmentation spectra were obtained.
  2. At least 1 annotation match in the mzVault, mzCloud or Chemspider Search.
To further improve the rigor of our discovery workflow, we performed additional manual filtering based on the following criteria:

  • Features with retention time earlier than 3 minutes on this HILIC column, which are nonpolar and should be quantified by a C18 column, were removed.
  • Features with predicted compositions containing chemical elements rarely found in human metabolome (e.g. certain halogens) were removed.
  • Features enriched in the lysosomes from only one independent experiment were removed.
Rigorous quantification of metabolite abundance was performed by TraceFinder (Thermo Fisher Scientific) in conjunction with an in-house library of known metabolite standards (MSMLS, Sigma-Aldrich). Isotopically labelled amino acids were used as internal standards. Mass tolerance for extracting ion chromatograms, 5 ppm.


Note
For LC wellness:

  • Make sure to transfer both WC and Lyso-IP samples from the original harvesting tubes to another NEW SET OF TUBES.
  • When taking the supernatant from the Lyso-IP sample, USE A MAGNET TO PREVENT DRAWING UP BEADS.