The unknown lipids project: harmonized methods improve compound identification and data reproducibility in an...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Metabolomics

Background:

• Untargeted lipidomics offers broad lipid analysis and discovery of novel compounds.
• Previous studies focused on targeted lipidomics reproducibility, leaving untargeted methods' inter-laboratory comparison unexplored.

Approach:

• Five laboratories analyzed reference samples using individual and a common untargeted lipidomics method.
• A standardized chromatographic and data analysis protocol was implemented.
• Lipid identification was performed using spectral search against the LipidBlast library.

Key Points:

• The common method enhanced chromatographic reproducibility and increased shared lipid feature detection between laboratories.
• Spectral search identified over 2,000 unique lipids.
• LC-MS/MS and ion mobility data analysis, including spectral networking, aided in classifying unknown lipid features.

Conclusions:

• Harmonized untargeted lipidomics methods improve inter-site reproducibility for lipid quantitation and feature alignment.
• This approach offers superior compound identification compared to targeted lipidomics.
• The developed method serves as a valuable reference for future lipidomics data annotation.