Structural Biochemistry/Lipids/Sample Preparation for Mass Spectrometry-Based Lipidomics Studies

Metabolomics is the study of nonproteinaceous, low molecular weight intermediates (metabolome) in the cell and the fats (lipids) constitute the largest subset in the metabolome. Hence, lipidomics is a subcategory of metabolomics and it is the detailed analysis and characterization of the structure and function of lipids. There are two types of lipidomics: targeted and untargeted. Targeted lipidomics is where the lipid species to be monitored is known before starting the analysis and untargeted lipidomics is simply exploring and trying to find new lipids. For untargeted lipidomics, mass spectrometry is used and searched for new m/z ratio peaks. A large variety of lipids constitute the cellular membranes, and analyzing their interactions with membrane-associating proteins can lead to further analysis, such as insight on drug interactions.

Sir JJ Thomson invented the mass spectrometer, which observes the mass-to-charge ratio of the sample. According to the article, "Applications of Mass Spectrometry to Lipids and Membranes," there are four main applications of mass spectrometry to the field of lipidomics (1): 1) Two current mass spectrometry-based lipidomics approaches 2) Mass spec-based structure determination of novel lipids 3) Deuterium exchange mass spectrometry to study the location and orientation of proteins associated with lipid membranes 4) Advances in the imaging of lipids in tissue through Matrix Assisted Laser Desorption mass spectrometry (1)

The LIPIDS MAPS Consortium has defined 8 categories of lipids based on their chemically function backbone: fatty acyls, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides

There are two methods to create sample preparations for mass spectrometry. The first method is comprehensive lipidomics analysis by separation simplification, which is better known as CLASS. This method is based on separation of different lipid categories using extraction and chromotographic separation. First, the components of the lipid are chromatographically separated and then added directly into the mass spectrometry ("divide-and-conquer" approach). To separate the components of the fatty acids, there are two methods, high powered liquid chromotagraphy and gas chromotography. HPLC couples directly into the mass spectrometer and "gas chromatography requires derivataization of the free fatty acid" since no electrospray ionization is in it (1). Electrospray ionization allows for the ionization of proteins in a liquid medium without derivitization. A small amount of ammonium acetate can be added to the electrospray ionization solution in order to increase the ionization efficiency. The CLASS approach minimizes ion suppression observed in the other method, shotgun approach. Shotgun approach omits chromatographic separation and analyzes all the lipid classes together while employing different ion source polarities. For this method, the researcher must carefully select electrospray ionization, electrospray ionization additives, and mass spectrometry mode. The downfall to this method is ion suppression. The low level species are not detected; therefore, the CLASS approach is a more preferred method.

For both methods, a general cell or tissue preparation is needed (1): 1) Cells or tissues cultured and subjected to a probe (activation or perturbation) 2) Lipidome is compared to a control unperturbed sample 3) Lipids stored within cell wall need to be disturbed through sonication and produce a uniform homogenate 4) Prior to extraction, use internal standard to enable absolute quantity of lipids in the sample 5) Solvent containing extracts evaporated and remaining lipids resuspended in liquid medium optimal for direct infusion into the mass spectrometer or in a medium compatible with either gas or liquid chromatography prior to CLASS (1)

References

1. Annu Rev Biochem. 2011 Jun 7;80:301-25. Applications of mass spectrometry to lipids and membranes. Harkewicz R, Dennis EA. Source Department of Chemistry and Biochemistry and Department of Pharmacology, School of Medicine, University of California at San Diego, La Jolla, California 92093-0601, USA. rharkewicz@ucsd.edu