Lipidomics Uses Light Touch to Distinguish Unsaturated Isomers

February 23, 2016

Lipidomics, the large-scale analysis of cellular lipids, is not only enriching the discipline of systems biology, it is also contributing to the development of new biomarkers. Yet for all the structural and mechanistic insights it is capable of providing, lipidomics continues to struggle with a blind spot. It has trouble distinguishing unsaturated lipid isomers, lipid structures that differ only in the locations of carbon-carbon double (C=C) bonds.

Without C=C bond information, lipidomic profiles are blinkered. They are liable to miss tissue-specific isomer differences, or they risk overlooking shifts in isomer populations that occur as diseases progress.

To help lipidomics see further, researchers based at Purdue University have refined a technique that has already done so much for the discipline. The technique, tandem mass spectroscopy, copes with the enormous diversity of lipids in two stages. In the first stage, lipid species are ionized and separated by a round of mass spectrometry on the basis of their mass/charge ratios. In the second stage, lipid species of a selected mass/charge ratio are broken into charged fragments, and these charged fragments are separated by another round of mass spectrometry.

As sensitive as this two-stage approach has proved to be, it does not, in general, distinguish between unsaturated lipid isomers. Workarounds do exist, but they require either large sample sizes or specialized equipment.

Another workaround, however, needs only small samples and readily available equipment. This alternative workaround, say the Purdue researchers who created it, can easily pinpoint the location of C=C bonds in lipid molecules, a capability that could lead to the early diagnosis of cancer.

The new approach appeared February 22 in the Proceedings of the National Academy of Sciences, in an article entitled “Identification and quantitation of lipid C=C location isomers: A shotgun lipidomics approach enabled by photochemical reaction.” The article described how the Purdue researchers used a photochemical reaction, the Paternò-Büchi reaction, to modify the C=C bonds into rings that could be easily cleaved into two parts. The resulting fragments, called C=C diagnostic ions, can be measured and identified during tandem mass spectrometry analysis.

Compared to other chemical derivatization methods the Paternò-Büchi reaction has several advantages, including fast reaction kinetics suitable for on-line coupling with ionization, wide applicability to different lipid classes, simplicity of implementation, and compatibility with commercial mass spectrometry MS instruments with lower energy collision-induced dissociation capability.

“The potential of this method has been demonstrated with an implementation into shotgun lipid analysis of animal tissues,” wrote the authors of the PNAS article. “Among 96 of the unsaturated fatty acids and glycerophospholipids identified from rat brain tissue, 50% of them were found as mixtures of C=C location isomers; for the first time, to our knowledge, the quantitative information of lipid C=C isomers from a broad range of classes was obtained. This method also enabled facile cross-tissue examinations, which revealed significant changes in C=C location isomer compositions of a series of fatty acids and glycerophospholipid species between the normal and cancerous tissues.”

Essentially, the system was demonstrated with brain tissue and breast cancer tissue from mice. The researchers, who also used the method to study liver and kidney tissue, indicated that they plan to include prostate cancer tissue in future research.

According to the researchers, the new method has two primary benefits. "One is as a new tool for biologists, to relate the isomeric ratio to the system biology and cell biology," said Zheng Ouyang, Ph.D., a study co-author and a professor of biomedical engineering, chemistry, and electrical and computer engineering at Purdue. "The second is to identify biomarkers for fast diagnosis of disease."

The method can be completed within hours, starting with small amounts of tissue—tens of milligrams—compared to weeks and hundreds of milligrams using conventional analytical techniques.

"More importantly, traditionally there has not been a good method for quickly distinguishing the unsaturated lipid isomers regardless of the amounts of tissue used," commented Dr. Ouyang. "We believe that the research community may now use this approach to discover biomarkers and eventually apply it to diagnosis."