Test files

"How to" Guide

What is required?

  • Quadrupole Orbitrap mass spectrometer set up for shotgun lipidomics
  • Internal standard mix: UltimateSPLASH™ ONE (Avanti Polar; cat. Nr.330820L-1EA)
  • Infusion spray solvent. We use the SprayMix: IPA/MeOH/CHCl3 4:2:1 v/v/v + 7.5 mM ammonium formate.
  • Xcalibur software (Thermo Fisher Scientific; a version supplied with your instrument).

1. Internal Standard preparation

Suggested dilution for Q-Exactive Orbitrap. Adapt the dilution based on the performance of your instrument -

  • Add 1 mL of SprayMix solvent to 10 µL of internal standard (UltimateSPLASH™ ONE)
  • Further dilute 100-fold in the sample plate compatible with shotgun analyses with SprayMix. If you use syringe infusion with a higher (~ μL/min) flow rate you might want to adjust the dilution ratio such that the intensity of most abundant peaks in MS1 spectra is close to 1×106 for positive and 1× 105 for negative mode.

2. Spectra acquisition

  • Once the spray stabilises, acquire: MS1 positive for ca. 1 min; MS2 for ca. 30 sec; MS1 negative ca. 1 min and MS2 negative for ca. 30 sec. Altogether, it should be around 3 min; however, the exact timing is not required (See Fig.1 as example). Use profile mode for all spectra.
  • Acquire MS positive within the mass range of m/z 150-1000; MS2 positive only for PC-d5 33:1 (m/z 751.6008); MS negative within the range m/z 150-1000; MS2 negative only for PE-d5 33:1 (m/z 707.5393). When calculating MS2 quality index we rely on monoisotopic peaks and (currently) neglect the first and second isotopes transmission because labs use different settings.
  • When acquiring MS2 spectra, apply stepped NCE at 10% and 30%, ideally dividing the acquisition time equally between the two NCE. The merged MS2 spectra should contain both precursor and fragment ions – this is necessary to benchmark the collisional fragmentation efficacy as explained in Home page.
Figure 1
Figure 1. The figure displays a representative spectrum collected in both positive and negative ion modes. Initially, the instrument acquires an unfragmented (full scan) spectrum followed by a targeted fragmentation event, applied within a specified m/z range and time window. An ideal TIC intensity should be around 1×108 - 1×109.


3. Peak list export

  • Open the acquired .raw file in Xcalibur software; it should look approximately like Figure 1.
  • Enable the “Resolution” and “All peaks” at the Display Options panel of Xcalibur (Figure 2A).
  • Select the ranges of stable spray for each mode (MS1 or MS2) and polarity (pos or neg); there should be four time ranges in total. It is not necessary to always select the same time range, but we recommend it should be about 1 min for each MS1 spectrum and, at least, 30 sec for MS2 spectra. The duration of time ranges should support representative averaging of peaks.
  • Export peak lists annotated with m/z (Exact Mass), Intensity, and Resolution into the clipboard (Figure. 2B) and copy them into individual spreadsheets in the same Excel file. The order must reflect that shown in Figure. 2C.
Figure 2A
Figure 2A. Guide to enable the export of the resolution.
Figure 2B
Figure 2B. Guide to enable the export of all peaks.
Figure 2C. Example of an import file needed for the QC Shotgun software. Highlighted in red are the important details to consider for the correct use of the software.


4. Single file import

Single file import Single file import Single file import Single file import



5. Multiple files import

Multiple files import Multiple files import Multiple files import



6. Reference range of values

Parameter Positive/Negative ion mode
Mass Accuracy ± 0-1.5 ppm
Resolution drop 15-20%
Isotopic Concordance 95-100%
Class Response 1-10 avg RSD
In-source Fragmentation <0.1 – 5%
Sensitivity 5-25%
MS2 Fragmentation 0-2



Contacts

Dr. Silvia Radrezza, radrezza@mpi-cbg.de