Cross-Laboratory Standardization of Preclinical Lipidomics Using Differential Mobility Spectrometry and Multiple Reaction Monitoring

Abstract

Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics’ technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950–Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231–Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

‘Omic-scale quantitative HILIC-MS/MS approach for circulatory lipid phenotyping in clinical research

“…Importantly, the presented HILIC approach offers the extended coverage of SP and GP species (along with ether-linked species, PI, PS, LPS, and LPS). For comparison, the commonly applied DIMS quantification method using Lipidyzer 18 and one of the most comprehensive RPLC based approaches 4 allow for detecting 737 and 636 unique lipid species in human plasma, respectively. In general, the coverage is dependent on the sample type and its composition 46 .…”

“…The final method in positive mode (for human plasma) has a significantly higher coverage (541 SRM) and therefore lower dwell times compared to independent methods during the initial screen (maximum 309 SRM). As specified above, the extensive coverage of our method is in accordance with the results reported with the Lipidyzer kit, where 704 lipid species belonging to 13 major lipid classes, among which more than 70% TG, were measured with high reproducibility (CV < 20%) in human plasma 18 . Using the presented HILIC approach, the coverage of SP and PL was significantly extended with TG comprising 49% of quantified lipid species.…”

“…The translation of lipid profiling to clinical laboratories is still hindered by lack of the necessary amount of cross-comparable quantitative lipid data acquired on different populations worldwide 14,15 . However, the fundamental efforts to establish analytical consensus values in reference materials (e.g., NIST SRM lipidomics community (http://lipidomics-standards-initiative.org) 3,16,17,18,19,20 . The systematic analysis of shared reference materials has emerged as the best normalization strategy to correct method-specific biases (e.g., application of different extraction procedures and analytical platforms for lipid measurement) 21,22,23,24,25 .…”

“…The method precision was evaluated through intra-and inter-day analysis of independently extracted pooled plasma samples and NIST reference material as quality control. Lipid concentration ranges were measured in different NIST plasma reference materials (SRM 1950, Diabetic -T1D, High triglyceride -h-TG, Young African-American -YAA) and cross-evaluated against previous inter-laboratory studies 18 . Finally, the established HILIC-MS/MS method was applied to a sub-group of clinically healthy, aged participants from the COmPLETE-Health study to elucidate sex differences across human lipid profiles.…”

Deep Metabolic Profiling Assessment of Tissue Extraction Protocols for Three Model Organisms

“…Although such monophasic extractions are in general well suited for lipidomic approaches, the broad nature of the standardized metabolic profiling assay requires a trade-off between coverage, extracted concentration as well as reproducibility across all metabolite classes. The latter criterion was recently highlighted by Ghorasaini et al (2021) in an interlaboratory assessment of extraction protocols for lipidomic analyses. The authors showed that the extraction with MeOH/MTBE performed better and was more practical than the comparable Bligh and Dyer extraction.…”