New Frontiers for Mass Spectrometry in Lipidomics, Part II

Jun 01, 2012
Volume 30, Issue 6, pg 482–491

Innovative technologies are rapidly facilitating our ability to measure the numerous and diverse lipids present in biological samples. In the second and final installment of this series, we present and discuss the use of those technologies for lipid analysis.

In part I of this series, we introduced the concept of lipidomics as a new research strategy for analyzing lipids using state-of-the-art mass spectrometry (MS) technology. Together with genomics, transcriptomics, and proteomics, lipidomics is now routinely used in various scientific areas including drug and biomarker discovery, drug development, nutrition, biotechnology, and metabolic engineering research. Moreover, the future of lipidomics holds significant promise to further the development of personalized medicine.

Researchers use lipidomics as a tool to analyze a selected set of lipids (targeted lipidomics). They also use it, in a more comprehensive way, to screen all lipids present in a biological sample (untargeted lipidomics). A lipidomic research strategy presupposes the choice of analytical solutions — sample preparation, chromatography, ionization sources, and MS analyzers — that are more appropriate for answering the biological questions posed. In the second and final installment of this series, we present and discuss the use of those technologies for lipid analysis.

Sample Preparation

The high sensitivity of MS offers less labor-intensive lipid analyses. Nevertheless, the quality of sample preparation remains an important factor for success. Sample preparation includes lipid extraction from the biological matrix and elimination of nonlipid contaminants. Usually, lipid concentrations in biological samples are normalized by volume, tissue weight, cell number, or protein or DNA concentration. Quantification is done by lipids labeled with stable isotopes or nonendogenous structural analogs (internal standards) that are added before the extraction procedure. The methods for lipid extraction vary according to the sample matrix, the particular lipids under investigation, and the subsequent analytical method. The most common methods for lipid extraction are liquid–liquid extraction and solid-phase extraction (SPE).

Liquid–Liquid Extraction

Jordi Folch and colleagues (1) and E.G. Bligh and W.J. Dyer (2) developed the most widely accepted methods for lipid extraction from biological samples. Those methods are based on chloroform–methanol protocols that include phase partitioning of lipids into the organic layer. For a large variety of physiologically relevant lipids, the protocols work relatively well, and they have been adapted toward recovering more complex lipid chemistries as well as low-abundance lipid species. Multistep extractions and pH alterations also can be used for recovering acidic phospholipids such as gangliosides and phosphoinositides. Alternative extraction procedures that use less toxic organic solvents such as methyl-tert-butyl ether, hexane–isopropanol, and ethyl acetate–ethanol mixtures have been proposed for a wide range of tissues.

Solid-Phase Extraction

SPE has become a frequently used analytical procedure for rapid, preparative separation of total lipid extracts into different lipid classes. SPE involves using diverse solvents and commercial, prepacked columns with various stationary phases including silica and reversed-phase materials. (The latter use chemically bonded octadecylsilyl [ODS, C18] groups or ion-exchange media such as those with bonded aminopropyl or phenylsulfonic acid moieties.) Lipid extraction from a complex matrix might be achieved by choosing conditions (solvents and stationary phases) so that the required analyte is retained on the column while the impurities pass directly through. Conversely, extraction occurs by allowing the analyte to be eluted through the column while the impurities are retained. Targeted lipidomic studies often use SPE for fractionating the low-abundance lipids species from the highly abundant species like phospholipids, creating dynamic-range and ion-suppression issues.

Lipidomics Approaches

These three MS-based lipidomics approaches are currently in use:

  • Direct infusion MS (for example, shotgun lipidomics)
  • On-line chromatographic separation-MS (for example, liquid chromatography–mass spectrometry [LC–MS])
  • Surface-based desorption ionization MS (for example, MS imaging)

Ion mobility spectrometry, a post-ionization separation technique, can be coupled to any of these approaches.

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