Apr 20, 2026

Metabolomic analysis of polyamines

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gp9z7dvzp/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815;
  • 3Laboratory of Applied Mass Spectrometry, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium;
  • 4Metabolomics Core Facility Leuven, Center for Cancer Biology, VIB, Leuven, Belgium
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gp9z7dvzp/v1
Protocol Citation: Sarah van Veen, Peter Vangheluwe, Bart Ghesquiere, Sam De Craemer 2026. Metabolomic analysis of polyamines. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9z7dvzp/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: December 13, 2024
Last Modified: April 26, 2026
Protocol  Integer ID: 115161
Keywords: ASAPCRN, metabolomics, polyamines, metabolomic analysis of polyamine, analysis techniques for polyamine profiling, polyamine profiling, metabolomic analysis, mass spectrometry analysis of metabolite, polyamine, metabolite, mass spectrometry analysis, cerebrospinal fluid, astrocyte, brain tissue
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol details the extraction, derivatization, and mass spectrometry analysis of metabolites from cells, astrocyte-conditioned medium (ACM), plasma, cerebrospinal fluid (CSF), and brain tissue. It includes steps for preparing samples of different types and volumes, LC-MS settings, and data analysis techniques for polyamine profiling.
Materials
Materials
  • NaCl (Cat# 0962.2; Carl Roth)
  • Trichloroacetic Acid (TCA; Cat# T9159; Sigma)
  • Sodium carbonate (Cat# S7795-500g; Sigma)
  • Isobutyl chloroformate (Cat# 177989-25G; Sigma)
  • Diethyl ether (Cat# 309966-1L; Sigma)
  • Acetonitrile (Cat# 83640.320; VWR)
  • Formic acid (Cat# 84865.260; VWR)
  • 1,6-Hexanediamine (Cat# H11696; Sigma)
  • 13C5 ornithine (Cat# CLM-4724-H-PK; Cambridge Isotope Laboratories, Inc)
  • 13C4 putrescine (Cat# CLM-6574-PK; Cambridge Isotope Laboratories, Inc)
  • 13C4 spermidine (Cat# CLM-9435-PK; Cambridge Isotope Laboratories, Inc)
  • D8 spermine (Cat# DLM-9262-PK; Cambridge Isotope Laboratories, Inc)

Equipment
  • Cell scraper
  • Eppendorf tubes (2 mL)
  • Centrifuge
  • Vacuum centrifuge
  • LC-MS system (Vanquish LC coupled to Q Exactive Orbitrap Focus, Thermo Scientific)
  • ACQUITY UPLC HSS C18 column (1.7 μm, 2.1 x 100 mm, WATERS)
  • ACQUITY UPLC HSS T3 VanGuard Pre-column (100Å, 1.8 µm, 2.1 mm x 5 mm, WATERS)
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Metabolomic analysis of cells
1h 35m
Sample preparation
Wash cells with 0.9% NaCl.
Add 150 µL of ice-cold 6% TCA to extract metabolites.

Collect lysate with a cell scraper and incubate On ice for 00:30:00 .

30m
Centrifuge lysate at 20000 x g, 4°C, 00:20:00 .

20m
Collect the supernatant, flash-freeze in liquid nitrogen, and store at -80 °C .

Derivatization
Add 900 µL of sodium carbonate buffer to 100 µL of supernatant.

Add 25 µL of isobutyl chloroformate, incubate at 35 °C for 00:30:00 .

30m
Transfer 800 µL of the reaction mixture to a 2 mL Eppendorf tube.

Add 1 mL of diethyl ether, vortex, and incubate for 00:15:00 at 25 °C .

15m
Collect 900 µL of the upper phase, dry under vacuum, and dissolve in 125 µL of 50% acetonitrile in water with 0.1% formic acid.

Transfer to an MS vial.
LC-MS analysis
Load 10 µL onto the LC system with an ACQUITY UPLC HSS C18 column.

Use solvent A (H2O with 0.2% acetic acid) and solvent B (acetonitrile with 0.2% acetic acid).
Apply a gradient:
- 0–2 min: 20% B
- 2–10 min: Linear increase to 85% B
- 10–17 min: Hold at 85% B
- 18 min: Return to 20% B
Maintain flow rate at 250 μL/min and column temperature at 30 °C .
Set mass spectrometer to full scan (70–750 m/z), positive mode.
Data analysis
Integrate peak areas using El-Maven (Elucidata).
Metabolomic analysis of ACM, plasma, CSF and brain tissue
30m
Sample preparation - biological fluids and tissue
Mix 50 µL of ACM or plasma with 50 µL of ice-cold TCA extraction buffer containing labeled internal standards.

Note
Internal standards: 13C5 ornithine (Cat# CLM-4724-H-PK), 13C4 putrescine (Cat# CLM-6574-PK), 13C4 spermidine (Cat# CLM-9435-PK) and D8 spermine (Cat# DLM-9262-PK) from Cambridge Isotope Laboratories, Inc.

For cerebrospinal fluid (CSF), combine each sample with an equal volume of ice-cold 6% TCA extraction buffer containing labeled internal standards.
Note
Due to the limited volume obtained per animal, a subset of CSF samples may need to be pooled prior to extraction to ensure sufficient material for LC-MS/MS analysis. Pool samples across animals of the same genotype and sex. Pooled samples are processed identically to individual samples. In figures, represent individual samples as filled symbols and pooled samples as open symbols.

For cortical brain tissue (15–30 mg), add 250 µL of ice-cold TCA extraction buffer containing labeled internal standards and homogenize samples in Ribolyser tubes to ensure thorough extraction.
Centrifuge and collect 40 µL of supernatant for derivatization.

Derivatization
Add 360 µL of sodium carbonate buffer and 10 µL of isobutyl chloroformate.

Incubate at 35 °C for 00:30:00 .

30m
Transfer to an MS vial.
LC-MS analysis
Load 50 µL onto the LC system with a VanGuard Pre-column.
Apply a gradient:
- 0–0.67 min: 5% B, flow rate 1.8 mL/min to waste
- 0.67–1 min: 5–35% B
- 1–1.1 min: Flow reduced to 0.3 mL/min, increase to 36% B
- 1.1–3 min: Linear increase to 85% B, hold until 4.7 min
- 4.7–4.73 min: Redirect flow to waste, decrease flow to 1.8 mL/min
- 4.73–4.86 min: Decrease to 5% B, equilibrate
Maintain column temperature at 25 °C .

Data analysis
Integrate peak areas using El-Maven (Elucidata).