May 30, 2024

Public workspaceBAF_Protocol_010 Metabolomics LC-MS(/MS): Vanquish UPLC and Orbitrap ID-X

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l629ddgqe/v1
  • Nicholas Sherman1
  • 1University of Virginia Biomolecular Analysis Facility Core
Nicholas Sherman
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l629ddgqe/v1
Protocol Citation: Nicholas Sherman 2024. BAF_Protocol_010 Metabolomics LC-MS(/MS): Vanquish UPLC and Orbitrap ID-X. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l629ddgqe/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: May 15, 2024
Last Modified: May 05, 2025
Protocol Integer ID: 99848
Keywords: mass spectrometry, metabolomics, LC, instrument parameters, baf-protocol-010 metabolomics lc, parameters for metabolite run, metabolite run, proteomic, basic lc, proteomics so the gradient, m, orbitrap id, vanquish uplc
Abstract
This protocol is the basic LC and MS running parameters for metabolite runs. The samples typically have fewer components (ions) than proteomics so the gradients are shorter and most molecules are singly charged.
Guidelines
These are our general settings as a starting point for untargeted metabolomics experiments. Specific adjustments need to be made to meet specific samples or type of data required.
Materials
Thermo Orbitrap ID-X - FETD1-10001
Thermo Vanquish Duo UHPLC
Waters ACQUITY UHPLC BEH C18 1.7 um, 2.1 x 150 mm - 186002353
Thermo Optima 0.1% FA (formic acid) in water -  LS118-4
Thermo Optima Methanol - A456-212
Thermo Autosampler vials 0.25 mL – 14-823-136
Thermo Orange caps – 14-823-380
Fisherbrand Standard Pipette Tips (200 uL - Yellow) – 53503-065
Cambridge Isotope Laboratories QRESS heavy labeled standards - MSK-QRESS-KIT
Prepare samples for injection
Suspend dried metabolite extracted samples with Amount100 µL of 0.1% Formic acid in water containing 100X diluted Metabolomics QRESS heavy labeled standards.

Vortex, microfuge for Duration00:15:00 at max speed, and take off Amount50 µL into autosampler vial (do not touch bottom of tube).

15m
Make sure there is no air in the bottom of the vial, carefully add the vial into the autosampler of the UPLC Vanquish system.
In the sequence setup view of the Xcalibur software include each sample in a row filling with: file name, sample ID, folder directory to save results, directory for the acquisition method, vial position and injection volume. Normally use 20% of sample.
LC parameters 15 min gradient
Speed limits: draw speed 1 uL/s; dispense speed 1 uL/s.
Injection wash parameters: Wash mode: both; Wash time: 10.0s; Wash speed: 10.0 uL/s. Column temperature: Temperature30 °C
Run time: Duration00:15:00
Solvents: %A1: Water, 0.1%FA.
    %A2: Methanol, 0.1% FA.
Upper limit pressure: 1500 bar
Flow = 0.250 ml/min
Equilibration: -3min, 0% B, curve = 5.
Gradient (all steps curve = 5): 0 min 5%B; 8 min 50% B; 8-9 min 98% B; 9-13 98% B;  13-13.1 min 0%B; 13.1-15 min 95%B.
15m
General Instrument (Orbitrap ID-X) – Positive and Negative modes
Ion Source Type: H-ESI
Spray Voltage Positive 3500V
Spray Voltage Negative 2500V
Ion Transfer Tube Temperature Temperature275 °C , Sheath gas 35, Aux Gas 7, Sweep gas 2, Vaporizer Temp Temperature320 °C
Default charge state: 1
Full scan parameters
Scan 67-1000 m/z, resolution 60K, Quadrupole isolation: True, normalized AGC 25%, RF lens 35%, Max. Inj. Time 50ms, 1 microscan, Data Type Profile, Polarity Positive or Negative (create two methods one for Positive acquistion and another for Negative acquisition)
MS/MS parameters
Min intensity 2E4, Dynamic exclusion - 1 time, 2.5s, 10ppm, isolation 1.5 m/z, resolution 15K, HCD NCE stepped 20, 35, 50%, Max. Inj. Time 25ms, normalized AGC 25%, 1 microscan, Data Type Centroid.