Comprehensive UHPLC–MS/MS Lipidomics Profiling to Study the Effects of Betulin on Keratinocytes

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The Column spoke to Carlos Calderón about his research project, investigating pharmacolipidomics analysis of betulin in human primary keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin.

Q. How did the idea of this research project arise and what are the aims of this research?

A: Our research collaboration partner, Irmgard Merfort from the University of Freiburg, Germany, has previously investigated the bioactivity of the natural compound betulin, a pentacyclic triterpene, which exerts a variety of different biological effects, including anti-cancer, anti-inflammatory, anti-fungal, and anti-viral activities. Studies have been conducted in her laboratory to explain the clinically proven wound-healing effects of betulin (1–3). Those studies found that betulin influences the inflammatory phase and the new-tissue-formation phase in the wound-healing process, which enhances keratinocytes migration and stimulates their differentiation.

In our laboratory, we decided to undertake a pharmacolipidomics analysis of betulin in human primary keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin.

Our goal was to clarify whether, or in which way, the lipophilic betulin may act on cell membranes, permeate membranes to enter the cell, or have an impact on the lipid profile of the cell.

Q. What are you doing in this research that is novel?

A: We performed a comprehensive description of the lipidome of human immortalized keratinocytes and their changes following treatment with betulin. The novel aspect of this research is the results detailing the changes in upregulation and downregulation of lipid species following betulin treatment. Additionally, for the identification of lipids we have employed a novel targeted data-processing approach combining the confidence given from the comparison of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) fragmentation patterns, with the confidence given from the analysis of reversed-phase liquid chromatography (LC) elution patterns of lipids belonging to the same lipid class. Most lipidomic studies are based on data‑dependent acquisition (DDA) which acquires MS data comprehensively but only MS/MS data from the most abundant signals and for structural confirmation only. This means that when an MS signal is above a certain threshold an MS/MS experiment is triggered. It provides high-quality spectra for identification but no comprehensive MS/MS data across the peak and chromatogram which means no extracted ion chromatograms (EICs) can be generated on the MS/MS level. In our study based on independent data acquisition (IDA), EICs can be extracted on an MS/MS level, and can also be used for fragment ions, which provides additional options for data processing, higher confidence, and assay specificity (4).

Q. What were the main analytical challenges you had to overcome?

A: Our main analytical challenge was the identification of lipid species in the keratinocytes. Our untargeted LC–MS approach with data independent acquisition (DIA) using sequential window acquisition of all theoretical mass spectra (SWATH) analysis generates large amounts of data. Tools for processing the data and performing identification of lipids are available. However, many misidentifications are also obtained. These can insert bias into the findings of the study. For this reason, the aforementioned targeted MS data-processing was employed.

Q. What were your main findings and why are they useful?

A: We made a detailed characterization of the lipidome in keratinocytes. At the same time, we showed that there are significant differences in keratinocytes after treatment with betulin. In total, 611 lipids were identified at the “lipid species level” and 440 of them were shown to be significantly changed. Changes can be described in terms of lipid classes. Cholesteryl esters and diacylglycerides were significantly down‑regulated in betulin-treated samples and glycerophospholipids, sphingolipids,
and diacylglycerides were up-regulated.

Additionally, it was observed that the presence of betulin in extracts of keratinocytes, which were previously treated with betulin and washed, indicated that betulin may be incorporated into the membrane of keratinocytes. Also, the presence of other triterpenes in samples treated with betulin and absent in control samples could represent betulin metabolized within the keratinocytes (4).

Q. How are you planning to develop this research further?

A: Based on the obtained results, we would like to develop more targeted experiments to determine how those changes in specific lipid classes following betulin treatment are related with the healing properties of the compound in the skin.

References

1. U. Woelfle, M.N. Laszczyk, M. Kraus, K. Leuner, A. Kersten, B. Simon-Haarhaus, A. Scheffler, S.F. Martin, W.E. Müller, D. Nashan, and C.M. Schempp, J. Invest. Dermatol. 130, 113–123 (2010).
2. S. Ebeling, K. Naumann, S. Pollok, T. Wardecki, S. Vidal-y-Sy, J.M. Nascimento, M. Boerries, G. Schmidt, J.M. Brandner, and I. Merfort, PLoS One9(1) e86147 (2014).
3. T. Wardecki, P. Werner, M. Thomas, M.F. Templin, G. Schmidt, J.M. Brandner, and I. Merfort, J. Nat. Prod.79, 1112–1123 (2016).
4. C. Calderón, L. Rubarth, M. Cebo, I. Merfort, and M. Lämmerhofer, Proteomics. 1900113 (2019).

Carlos Calderón won a poster award for this research at HPLC 2019 in Milan, when he was working at the University of Tubingen, Germany. He can be contacted at carloseduardo.calderon@ucr.ac.cr