The field of lipid analysis is moving swiftly towards using LC-MS/MS techniques as a more accurate and thorough means for absolute lipid quantification in biological matrices. Due to ease of use and their sensitivity, ELISAs have been routinely used over the past three decades to quantify lipids. However, they do have limitations when looking to investigate multiple analytes, analyze small sample sizes, or work with complex matrices. Advances in mass spectrometry technology and software have helped to overcome many of the limitations of ELISAs. The techniques involving LC-MS/MS are now well established and offer the ability to accommodate numerous lipids in one analysis. Because many researchers who may be interested in LC-MS/MS analysis do not have the equipment or the expertise to perform it, Cayman offers lipidomic analysis as a contract service for customers. Our analysis service offers the sensitivity, efficiency, and specificity needed to identify and quantify eicosanoids and other oxylipins, ceramides and other sphingolipids, phospholipids, cholesterol esters, glycerolipids, and short-chain fatty acids.
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Dr. Miguel Gijón, Ph.D., runs data analysis in Cayman’s mass spectrometry laboratory.
Cayman’s state-of-the art mass spectrometry laboratories are equipped with several liquid chromatography- and gas chromatography-coupled systems, including triple quadrupole and high-resolution accurate mass Orbitrap mass spectrometers, enabling us to offer bioanalysis services customized for your specific needs. Your project is in the hands of experts who have many decades of collective experience in lipid synthesis, purification, and characterization. We sat down with one of those experts, Miguel Gijón, Ph.D., to talk about Cayman’s Lipidomic Profiling Services capabilities.
I trained as a biochemist at the Universidad Complutense of Madrid, Spain. I started working with mediators of inflammation, including the phospholipid platelet-activating factor, as a graduate student, and ever since I have worked in the field of lipid mediators, more than 25 years. I came to Cayman because of the excellent reputation of this company in helping develop tools for lipid mediator research. More importantly, I was very interested in the challenge of bringing my expertise in lipid analysis to help build new analytical capabilities within the company, so some of the exciting new analytical tools can become available to a wider field of scientists.
There is a clear common thread: Mass spectrometry-based analytical techniques are now well established and offer the possibility to analyze a wide variety of lipids, often dozens or even hundreds of analytes from a single sample. Many researchers, both in academia and industry, want to use these tools, but in many cases the instrumentation and the expertise to perform the analysis are not available. That is the reason they contact us, as a company with a long tradition in offering analytical tools for bioactive lipids.
Lipid profiling holds the promise of accelerating scientific discovery in many research fields. One obvious example is research in life sciences related to human disease, where it is apparent that a great number and variety of factors are at play, only a fraction of which are currently known. In a similar way to progress based on genomics, proteomics, and metabolomics, by casting wider nets to analyze lipids we improve our chances to identify new mediators, or groups of mediators, that are involved in physiological and pathological processes, shedding light on mechanisms of disease and offering potential targets for prevention or treatment.
We have begun with a set of standards for the analysis of oxylipins such as prostaglandins and leukotrienes, a natural fit for Cayman, and we have included many of the analytes that are best known in the relevant literature to be biologically active in a wide variety of models, particularly in promoting inflammation or the resolution of inflammation. The list of analytes can be easily customized according to each experiment, simply by modifying the list of mass-to-charge (m/z) transitions analyzed by the mass spectrometer and substituting the necessary internal and calibration standards. Cayman is starting to manufacture and distribute a new line of high-quality standards, labeled MaxSpec®, in which the concentration, purity, and stability are guaranteed, thus ensuring reproducible and accurate quantitation.
Our lab houses state-of-the art Triple quad and Orbitrap instruments, giving us the sensitivity, dynamic range, and analytical flexibility to provide qualitative (relative) and fully quantitative data to our customers in targeted experiments. If required we also have the instrumentation to generate high-resolution spectra, which is extremely useful for distinguishing between closely related isobaric molecular species, such as identification of unknown analytes in untargeted experiments.
We can handle any kind of biological sample, including cells in culture, biological fluids, or tissue samples. We recommend that samples be frozen as soon as possible and shipped frozen to us to minimize further metabolism or degradation of lipid mediators. Upon thawing, we make sure to immediately add the appropriate organic solvents, depending on the extraction procedure required, and a mix of the corresponding internal standards, which then become markers of any potential changes affecting the analytes during the extraction procedure.
As part of an ongoing collaboration, we recently received mouse skin samples, with which we had no previous experience and that are known to be rather challenging for quantitative analysis. We had to change our homogenization and extraction methods, but were able to obtain interesting, reproducible data on a variety of lipid mediators.
The first challenge is to ensure the correct identification and integration of the chromatographic peaks corresponding to the different analytes. Unfortunately, there is currently no software that can be relied upon to perform this task reliability, so Cayman has a qualified scientist skilled in mass spectrometry analysis of lipids perform this review for all projects. Peaks need to be reviewed manually, although certain visualization features do facilitate this task. Another major challenge is to analyze data for large numbers of analytes from large numbers of samples. This requires statistical tools such as principal component analysis (PCA) and volcano plots to reveal differences between experimental groups. When the complexity of the data requires it, we perform these types of analysis for our customers, and we are always open to discuss the results and help with the interpretation and generation of new hypotheses stemming from the lipid analysis.
I believe it is fair to state that Lipidomics is still in its infancy, so the potential for growth is immense. Improvements can be made in sensitivity, as many bioactive lipids are present at very low levels in biological samples. As of today, analysis of certain lipids requires prior separation from other lipids that are much more abundant and can completely mask the signals for the minor components. Developing extraction strategies that will allow for the analysis of different classes of molecules at the same time remains a major challenge. Another area of potential growth is the development of techniques that can be used in combination with current LC-MS or LC-MS/MS approaches to further define the molecular species analyzed (e.g., the position and configuration of double bonds in the acyl chains of phospholipids and glycerolipids). Finally, a critical feature of lipid mediators is that they are produced at specific locations within tissues and cells. Promising results in lipid mass spectrometry imaging will hopefully be expanded to add a spatial/compartmental dimension to the data, and eventually become one more technique in the Lipidomics toolbox.
For more information on Cayman’s Lipidomic Profiling Services download our scientific poster:
Learn more about our service capabilities at www.caymanchem.com/services.