Accurate Quantification of Lipid Species Affected by Isobaric Overlap in Fourier-Transform Mass Spectrometry

Authors: Regensburg University Hospital, European Molecular Biology Laboratory, Germany; University of Southern Denmark, Denmark


Lipidomics data require consideration of ions with near-identical masses, which comprises among others the Type-II isotopic overlap. This overlap occurs in series of lipid species differing only by number of double bonds (DBs) mainly because of the natural abundance of 13C-atoms. High-resolution mass spectrometry, such as Fourier-transform mass spectrometry (FTMS), is capable of resolving Type-II overlap depending on mass resolving power. In this work, we evaluated FTMS quantification accuracy of lipid species affected by Type-II overlap. Spike experiments with lipid species pairs of various lipid classes were analyzed by flow injection analysis-FTMS. Accuracy of quantification was evaluated without and with Type-II correction (using relative isotope abundance) as well as utilizing the first isotopic peak (M+1). Isobaric peaks, which were sufficiently resolved, were most accurate without Type-II correction. In cases of partially resolved peaks, we observed peak interference causing distortions in mass and intensity, which is a well-described phenomenon in FTMS. Concentrations of respective species were more accurate when calculated from M+1. Moreover, some minor species, affected by considerable Type-II overlap, could only be quantified by M+1. Unexpectedly, even completely unresolved peaks were substantially overcorrected by Type-II correction because of peak interference. The described method was validated including intraday and interday precisions for human serum and fibroblast samples. Taken together, our results show that accurate quantification of lipid species by FTMS requires resolution-depended data analysis.

Samples were infused using the Advion Interchim Scientific® TriVersa NanoMate®.

Hormone-Sensitive Lipase Couples Intergenerational Sterol Metabolism to Reproductive Success

Authors: University of Graz, BioTechMed-Graz, Austria; Max Planck Institute of Molecular Cell Biology and Genetics, Germany


Triacylglycerol (TG) and steryl ester (SE) lipid storage is a universal strategy to maintain organismal energy and membrane homeostasis. Cycles of building and mobilizing storage fat are fundamental in (re)distributing lipid substrates between tissues or to progress ontogenetic transitions. In this study, we show that Hormone-sensitive lipase (Hsl) specifically controls SE mobilization to initiate intergenerational sterol transfer in Drosophila melanogaster. Tissue-autonomous Hsl functions in the maternal fat body and germline coordinately prevent adult SE overstorage and maximize sterol allocation to embryos. While Hsl-deficiency is largely dispensable for normal development on sterol-rich diets, animals depend on adipocyte Hsl for optimal fecundity when dietary sterol becomes limiting. Notably, accumulation of SE but not of TG is a characteristic of Hsl-deficient cells across phyla including murine white adipocytes. In summary, we identified Hsl as an ancestral regulator of SE degradation, which improves intergenerational sterol transfer and reproductive success in flies.

Nano-ESI analysis was performed by chip-based infusion using the Advion Interchim Scientific® TriVersa NanoMate®.

Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of SNCA-A53T Human Dopamine Neurons

University of Cambridge, Royal Holloway University of London


  • Combining FACS and LESA-MS to establish high-throughput single cell lipid profiling
  • Lipid differences found within and between populations of human dopamine neurons
  • Inter-cell lipid heterogeneity is increased in SNCA-A53T dopamine neurons
  • Identification and isolation of human iPSC-dopamine neurons with a TH-RFP reporter


Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism, it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolution mass spectrometry we have developed a high-throughput method for untargeted single cell lipid profiling. This technological advance highlighted the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein (SNCA) mutant neurons have impaired membrane function. These results demonstrate that this single cell lipid profiling platform can provide robust data that will expand the frontiers in biomedical research.

Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

The brain is a remarkably complex organ and cholesterol homeostasis underpins brain function. It is known that cholesterol is not evenly distributed across different brain regions; however, the precise map of cholesterol metabolism in the brain remains unclear. If cholesterol metabolism is to be correlated with brain function it is essential to generate such a map. Here we describe an advanced mass spectrometry platform to reveal spatial cholesterol metabolism in situ at 400-µm spot diameter on 10-µm tissue slices from mouse brain. We mapped, not only cholesterol, but also other biologically active sterols arising from cholesterol turnover in both wild type and mice lacking cholesterol 24S-hydroxylase (CYP46A1), the major cholesterol metabolizing enzyme.

Oxidative modification of skin lipids by cold atmospheric plasma (CAP): A standardizable approach using RP-LC/MS2 and DI-ESI/MS2 – Using the TriVersa NanoMate

The Advion TriVersa NanoMate nanoelectrospray ionization technology was selected as the ion source for its long, stable spray capabilities ideal for lipid analysis.



Cold atmospheric plasma (CAP) is an emerging source for the locally defined delivery of reactive species, and its clinical potential has been identified in the control of inflammatory processes, such as acute and chronic wounds, or cancerous lesions. Lipids, due to their localization and chemical structure as ideal targets for oxidative species, are relevant modifiers of physiological processes. Human forehead lipids collected on a target were treated by an argon plasma jet and immediately analyzed by direct-infusion high-resolution tandem mass spectrometry (DI-MS2) or liquid chromatography-tandem MS (RP-LC/MS2). Subsequent data analysis was performed by LipidHunter (University of Leipzig), LipidXplorer (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden), and LipidSearch (TS). With either MS method, all major lipid classes of sebum lipids were detected. Significant differences regarding triacylglycerols (predominantly identified in RP-LC/MS2) and ceramides (predominantly identified in DI-MS2) indicate experimental- or approach-inherent distinctions. A CAP-driven oxidation of triacyclglycerols, ceramides, and cholesteryl esters was detected such as truncations and hydroperoxylations, but at a significantly lower extent than expected. Scavenging of reactive species due to naturally present antioxidants in the samples and the absence of a liquid interphase to allow reactive species deposition by the CAP will have contributed to the limited amount of oxidation products observed. In addition, limitations of the software’s capability of identifying unexpected oxidized lipids potentially led to an underestimation of the CAP impact on skin lipids, indicating a need for further software development. With respect to the clinical application of CAP, the result indicates that intact skin with its sebum/epidermal lipid overlay is well protected and that moderate treatment will yield limited (if any) functional consequences in the dermal tissue.

University of Cambridge, NIHR BCR Metabolomics and Lipidomics Facility

What is the focus of your lab’s research?

Almost exclusively high throughput lipid profiling of samples from large epidemiological studies, where we study gene-lifestyle interactions. The method works with plasma samples as well as dried blood spots. The method is also applied to small-scale studies of specific disease groups, dietary interventions and model systems such as yeast. The TriVersa NanoMate® is also used to study the lipid composition of tissues analyzed by LESA®.

Why did you incorporate the TriVersa NanoMate® into your laboratory?

The TriVersa NanoMate® is essential to efficiently analyze large-scale studies. It offers a really robust method for high throughput studies with minimal carryover.

Who would you recommend to purchase the TriVersa NanoMate®?

I recommend the TriVersa NanoMate® to laboratories with a large number of samples requiring a robust and reliable delivery system. The TriVersa NanoMate® eliminates typical nanoelectrospray ionization challenges.

Do you have any publications or presentations using the TriVersa NanoMate®?

Publication Highlight:
Development and Application of High-Throughput Single Cell Lipid Profiling: A Study of SNCA-A53T Human Dopamine Neurons
Snowden, et al. iScience, 2020, 23(10), 101703

Combining FACS and LESA-MS to establish high-throughput single cell lipid profiling. Research identifies lipid differences found within and between populations of human dopamine neurons.

Other Publications:
  • Snowden et al. Combining lipidomics and machine learning to measure clinical lipids in dried blood spots. Metabolomics. DOI: 10.1007/s11306-020-01703-0
  • Koulman et al. The development and validation of a fast and robust dried blood spot based lipid profiling method to study infant metabolism. Metabolomics. DOI: 10.1007/s11306-014-0628-z
  • Furse et al. A high-throughput platform for detailed lipidomic analysis of a range of mouse and human tissues. Analytical and Bioanalytical Chemistry. DOI: 10.1007/s00216-020-02511-0
  • Harshfield et al. An unbiased lipid phenotyping approach to study the genetic determinants of lipids and their associations with coronary heart disease risk factors. J Proteom Res. DOI: 10.1021/acs.jproteome.8b00786
  • Mann et al. Insights into genetic variants associated with NASH-fibrosis from metabolite profiling. Hum Mol Genet. DOI: 10.1093/hmg/ddaa162

Liquid Extraction Surface Analysis Mass Spectrometry (LESA-MS): Examples of a New Surface Probing Technique for Clinical and Pre-Clinical Applications

Executive Summary:

  • LESA and LESAplus liquid chromatography provide novel surface analysis tools for the spatial resolution of analytes from a wide variety of surfaces.
  • Whole body or organ sections in particular can be probed for small molecules, lipids and proteins with a resolution of 400 – 1000μm.
  • Commercial solution allows for automated processing of samples (Advion TriVersa NanoMate® LESA®).

This poster was presented at MSACL 2018 US in Palm Springs, CA.