Fast untargeted and targeted high-resolution mass spectrometry workflows for large-scale lipidomics

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Lipidomics is complex, requiring efficient high-throughput methods for large-scale analysis.
• Conventional hydrophilic interaction liquid chromatography (HILIC) coupled with untargeted acquisition is challenging due to complex data.
• A novel framework for untargeted data processing is needed to improve HILIC applicability.

Purpose of the Study:

• To present a detailed lipidomic workflow using fast HILIC separation.
• To establish a targeted high-resolution multiple reaction monitoring (MRMHR) method for lipid profiling.
• To evaluate the applicability of these workflows in large-scale clinical lipidomics.

Main Methods:

• A fast 6.7-min HILIC separation method was developed.
• Data-dependent acquisition/sequential window acquisition of all theoretical fragment ions analysis (DDA/SWATH) was employed.
• A targeted high-resolution multiple reaction monitoring (MRMHR) method was established for multi-class lipid profiling.
• Both workflows were applied to 240 case-control matched plasma samples.

Main Results:

• Lipid profiles of over 400 (targeted) and 500 (untargeted) features were generated.
• Lipid coverage differed between targeted and untargeted workflows at the species level.
• While individual sample quantitation varied, statistical comparisons of sample groups were largely concordant.
• Internal standard selection significantly influenced downstream statistical outcomes.

Conclusions:

• Fast HILIC-based workflows are applicable and reliable for high-throughput clinical lipidomics.
• These methods offer alternatives to longer separation techniques for untargeted and targeted analyses.
• Methodological considerations, including internal standard selection, are crucial for large-scale lipidomic studies.