Comprehensive and Reproducible Untargeted Lipidomic Workflow Using LC-QTOF Validated for Human Plasma Analysis

Abstract

The goal of this work was to develop a label free, comprehensive and reproducible high resolution LC-MS-based untargeted lipidomic workflow using a single instrument, which could be applied to biomarker discovery in both basic and clinical studies. For this, we have i) optimized lipid extraction and elution to enhance coverage of polar and non-polar lipids as well as resolution of their isomers, ii) ensure MS signal reproducibility and linearity, and iii) developed a bioinformatic pipeline to correct remaining biases. Workflow validation is reported for 48 replicates of a single human plasma sample: 1,124 reproducible LC-MS signals were extracted (median signal intensity RSD=10%), 50% of which are redundant due to adducts, dimers, in-source fragmentation, contaminations, or positive and negative ion duplicates. From the resulting 578 unique compounds, 428 lipids were identified by MS/MS, including acyl chain composition, of which 394 had RSD < 30% inside their linear intensity range, thereby enabling robust semi-quantitation. MS signal intensity spanned 4 orders of magnitude, covering 16 lipid subclasses. Finally, the power of our workflow is illustrated by a proof-of-concept study in which 100 samples from healthy human subjects were analyzed and the dataset investigated using three different statistical testing strategies in order to compare their capacity in identifying the impact of sex and age on circulating lipids.

Lipidomics unveils lipid dyshomeostasis and low circulating plasmalogens as biomarkers in a monogenic mitochondrial disorder

“…The coverage of lipid metabolites beyond mitochondrial β-oxidation was, however, limited. Thus, in the present study, we applied an untargeted lipidomic workflow using high-resolution LC-quadrupole time-of-flight (LC-QTOF), which has been previously validated and shown to detect more than 1,000 reproducible MS signals or features (defined by a mass-to-charge ratio [m/z], retention time, and signal intensity) in human plasma samples and cover 16 lipid subclasses (25).…”

“…Among all aforementioned lipids discriminating LSFC patients from controls, we focused on plasmalogens given that they are recognized hallmarks of primary peroxisomal defects (i.e., Zellweger and rhizomelic chondroplasia punctata syndromes; refs. 25,26). To further support our hypothesis of a contribution of peroxisomes to lipid dyshomeostasis in LSFC patients, additional targeted analyses were performed using LC-triple quadrupole (LC-QQQ).…”

“…Untargeted lipidomic analyses using LC-QTOF. Untargeted lipidomic analyses were performed using a previously validated workflow (25), which is herein briefly summarized. Lipids were extracted from plasma or liver after spiking with the following internal standards: LPC 13:0, PC19:0/19:0, PC14:0/14:0, PS12:0/12:0, PG15:0/15:0, and PE17:0/17:0 (Avanti Polar Lipids Inc).…”

“…The objectives of this study were to test the hypothesis that lipid dyshomeostasis in mitochondrial disorders goes beyond impaired mitochondrial FA β-oxidation, and may involve, among other disturbances, changes in peroxisomal lipid metabolism. To achieve our goals, we used both untargeted and targeted lipidomics based on liquid chromatography-mass spectrometry (LC-MS) (15,24,25). These analyses were conducted first in our homogeneous cohort of genetically defined LSFC patients and their prospectively matched healthy controls (12).…”

Reducing 14-3-3ζ expression influences adipocyte maturity and impairs function

“…Methanol fixed, siRNA-transfected, differentiated 3T3-L1 cells were processed for lipid extraction and untargeted LC-MS lipidomics, as previously described (20). In brief, samples (0.…”