Abstract
Standardization is essential in lipidomics and part of a huge community effort. However, with the still ongoing lack of reference materials, benchmarking quantification is hampered. Here, we propose traceable lipid class quantification as an important layer for the validation of quantitative lipidomics workflows. 31P nuclear magnetic resonance (NMR) and inductively coupled plasma (ICP)–mass spectrometry (MS) can use certified species-unspecific standards to validate shotgun or liquid chromatography (LC)-MS-based lipidomics approaches. We further introduce a novel lipid class quantification strategy based on lipid class separation and mass spectrometry using an all ion fragmentation (AIF) approach. Class-specific fragments, measured over a mass range typical for the lipid classes, are integrated to assess the lipid class concentration. The concept proved particularly interesting as low absolute limits of detection in the fmol range were achieved and LC-MS platforms are widely used in the field of lipidomics, while the accessibility of NMR and ICP-MS is limited. Using completely independent calibration strategies, the introduced validation scheme comprised the quantitative assessment of the complete phospholipid sub-ome, next to the individual lipid classes. Komagataella phaffii served as a prime example, showcasing mass balances and supporting the value of benchmarks for quantification at the lipid species level.
Highlights
To date, accurate absolute quantification remains a grand challenge in lipidomics.[1]
A certified standard of phosphoserine was used for internal standardization in 31P nuclear magnetic resonance (NMR)
Values from nine different methods on the lipid species, lipid class, or total PL content level were obtained. 31P NMR as a fully traceable method can be recommended for the quantitative assessment of unknown samples or sample pools in bigger cohort studies
Summary
Accurate absolute quantification remains a grand challenge in lipidomics.[1]. AIF is either used for the determination of the fatty acyl chain distribution of each class[40] or dedicated software solutions are necessary to resolve the chimeric spectra of DIA for lipid species quantification.[41] In this project, the disadvantage has been used as a benefit by integrating a class-specific fragment or a mass range typical for a lipid class over a certain retention time range. Figures of merit follow the EURACHEM guideline, The Fitness for Purpose of Analytical Methods, 2nd edition (2014).[46] MS-based LOD and limit of quantification (LOQ) were determined by multiplying the standard deviation of replicate (n = 5) injections of a low concentrated reference standard with 3 and 10, respectively. Standard deviation u2 (xi) and xi mean of technical replicates were used for calculation
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