Achieving Absolute Molar Lipid Concentrations: A Phospholipidomics Cross-Validation Study.

Area of Science:

• Lipidomics
• Analytical Chemistry
• Biochemistry

Background:

• Standardization is crucial in lipidomics, but a lack of reference materials hinders accurate quantification.
• Current validation methods for quantitative lipidomics workflows are limited.
• Traceable quantification of lipid classes is proposed as a key validation layer.

Purpose of the Study:

• To introduce and validate a novel method for traceable lipid class quantification.
• To establish a robust validation scheme for quantitative lipidomics.
• To demonstrate the utility of the proposed method using a real-world biological sample.

Main Methods:

• Utilized 31P nuclear magnetic resonance (NMR) and inductively coupled plasma-mass spectrometry (ICP-MS) with certified standards for initial validation.
• Developed a novel lipid class quantification strategy using lipid class separation and mass spectrometry with all-ion fragmentation (AIF).
• Applied independent calibration strategies to assess the complete phospholipid sub-ome and individual lipid classes.

Main Results:

• Achieved low absolute limits of detection in the femtomole (fmol) range using the AIF approach.
• Demonstrated the ability to quantitatively assess the complete phospholipid sub-ome.
• Successfully validated the lipidomics workflow using Komagataella phaffii, showing mass balances and supporting quantification at the lipid species level.

Conclusions:

• Traceable lipid class quantification provides a vital layer for validating lipidomics workflows.
• The novel AIF-based mass spectrometry approach offers high sensitivity and accuracy for lipid quantification.
• This method supports robust lipidomics data by enabling comprehensive mass balance assessments and species-level quantification.