Apr 28, 2025

Public workspaceLiquid Chromatography/Mass Spectrometry (LC/MS) based Untargeted Metabolite Analysis of Hydrolysate

DOI
dx.doi.org/10.17504/protocols.io.ewov1d8kkvr2/v1
  • Akila Wijerathna Yapa1,
  • Stanislav Sokolenko1
  • 1Department of Process Engineering and Applied Science, Dalhousie University, 5273 DaCosta Row, PO Box 15000, Halifax, B3H 4R2, NS, Canada
Akila Wijerathna Yapa
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov1d8kkvr2/v1
Protocol Citation: Akila Wijerathna Yapa, Stanislav Sokolenko 2025. Liquid Chromatography/Mass Spectrometry (LC/MS) based Untargeted Metabolite Analysis of Hydrolysate. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1d8kkvr2/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 15, 2024
Last Modified: April 28, 2025
Protocol Integer ID: 112169
Keywords: Liquid Chromatography, Mass Spectrometry, Untargeted Metabolomics, Hydrolysate, untargeted metabolite analysis, untargeted metabolomics profiling, extracted metabolite, standardized workflow for untargeted metabolomics profiling, metabolite extraction, reproducible metabolomic profiling, mass spectrometry, tandem mass spectrometry, reproducible metabolomic profiling for bioprocess, hydrolysate sample, using liquid chromatography, liquid chromatography, hydrolysate powder, defined chromatographic gradient, chromatographic gradient, hydrolysate, untargeted metabolite analysis of hydrolysate
Abstract
This protocol describes a standardized workflow for untargeted metabolomics profiling of hydrolysate samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hydrolysate powders were reconstituted in water, followed by metabolite extraction using a methanol-acetonitrile-formic acid solution with isotope-labeled internal standards. Extracted metabolites were separated using a Zorbax SB-CN analytical column under a defined chromatographic gradient and analyzed on a Q Exactive Orbitrap mass spectrometer operating in data-dependent acquisition (DDA) mode. MS1 scans were acquired at a resolution of 35,000, and MS2 scans were triggered for the top precursor ions. This method enables the comprehensive detection of polar and semi-polar small molecules in complex biological matrices and supports high-throughput, reproducible metabolomic profiling for bioprocess and biotechnology research applications.
Materials

General Materials

  • Deionized water
  • Microcentrifuge tubes (1.5 mL or 2.0 mL)
  • Vortex mixer
  • Pipettes (10 µL, 100 µL, 1000 µL) and pipette tips
  • Centrifuge capable of reaching 12,000 × g
  • SpeedVac centrifuge or vacuum concentrator


Reagents


Extraction solution:
  • Methanol
  • Acetonitrile
  • 1% formic acid in water (40:40:20 ratio)
  • Isotope-labeled amino acid standard (e.g., Cambridge Isotopes, CAT#MSK-A2)


Equipment for Mass Spectrometry

  • QExactive mass spectrometer
  • Vanquish liquid chromatography (LC) system
  • Analytical column: Zorbax SB-CN Rapid Resolution HD column (1.8 µm, 2.1 × 150 mm, Agilent)


Solvents for LC-MS

  • Mobile phase A: 0.1% formic acid in water
  • Mobile phase B: 0.1% formic acid in methanol


Consumables

  • LC-MS grade solvents (methanol, acetonitrile, formic acid, and water)
  • LC-MS vials with inserts


Miscellaneous

  • Freezer (-20°C) for sample incubation
  • Lab timer for incubation and centrifugation steps

Troubleshooting
Preparation of Powdered Hydrolysate
  1. Weigh the powdered hydrolysate sample.
  2. Reconstitute the powdered hydrolysate in deionized water to achieve a final concentration of 1 µg/µL.
  3. Vortex to ensure complete dissolution.
Metabolite Extraction
Resuspension Sampling:
  • Transfer 60 µL of the reconstituted hydrolysate solution into a microcentrifuge tube.
Add Extraction Solution:
  • Add 240 µL of the extraction solution (40:40:20 methanol/acetonitrile/1% formic acid) to the tube.
  • Include 3 µL of a 2X diluted isotope-labeled amino acid standard (e.g., Cambridge Isotopes, CAT#MSK-A2).
  • Drying:
  • Dry the collected supernatant in a SpeedVac centrifuge until completely desiccated.
Reconstitution:
  • Reconstitute the dried extract in 100 µL of deionized water.
Vortex to mix.
Sample Preparation for MS Analysis
Dilution:
  • Transfer 10 µL of the reconstituted sample to a vial suitable for mass spectrometry.
  • Dilute with 40 µL of deionized water.
Pooling Verification:
  • Ensure the dilution level aligns with prior analysis of the pooled sample to maintain consistency.

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis
Instrument Setup:
  • Use a QExactive mass spectrometer interfaced with a Vanquish liquid chromatography system.

Chromatographic Separation:
  • Employ a Zorbax SB-CN Rapid Resolution HD column (1.8 µm, 2.1 × 150 mm, Agilent).
  • Set the flow rate to 0.2 mL/min.
  • Apply the following gradient elution program:
  • Hold at 1% mobile phase B (0.1% formic acid in methanol) for 2 minutes.
  • Ramp to 15% B over 10 minutes.
  • Increase to 60% B in 3 minutes.
  • Ramp to 100% B in 4.9 minutes and hold for 2 minutes.
  • Return to 1% B in 0.1 minutes and hold for 5 minutes.
  • Mobile phase A: 0.1% formic acid in water.

Mass Spectrometry Settings:

MS1 Scans:
  • Resolution: 35,000
  • Automatic gain control (AGC)target: 3e6
  • Max injection time: 100 ms
  • Scan range: 75–900 m/z

MS2 Scans:
  • Resolution: 17,500
  • Automatic gain control (AGC) target: 1e5
  • Max injection time: 50 ms
  • Isolation window: 2 m/z
  • Loop count: 10
  • Normalized collision energy: 20 and 35
  • Scan range: 200–2000 m/z

Ion Source Parameters:
  • Positive ion spray voltage: 3,700 V
  • Negative ion spray voltage: -3,500 V
  • Capillary temperature: 300°C
  • Sheath gas: 45
  • Auxiliary gas: 15
  • Spare gas: 2
  • S-Lens RF level: 45
  • Probe heater temperature: 250°C

Acquisition Modes:
  • MS1: Profile mode.
  • MS2: Centroid mode.
Data Analysis
  1. Utilize a data-dependent acquisition scheme.
  2. Apply dynamic exclusion settings of 10 seconds to improve spectral quality.
  3. Export data for downstream processing and untargeted metabolomics analysis.
Acknowledgements
We would like to thank Dr. Christopher Hughes at the Biological Mass Spectrometry facility at Dalhousie University providing access to mass spectrometry and related technologies.