Core Technological Research and Development: Introduction to Lipid Mass Spectrometry
The two major classes of complex lipids in biological materials are glycerolipids and sphingolipids, which are substituted derivatives of glycerol and of sphingosine or a related long-chain base respectively. We have developed approaches involving predominantly electrospray ionization (ESI) and tandem mass spectrometry (MSn) on tandem quadrupole and quadrupole linear ion trap instruments to characterize both glycerolipids and sphingolipids, and among the biomedically important areas to which we have applied these approaches is the characterization of the complex lipids of pathogenic microorganisms, particularly with respect to differences in lipid synthetic pathways in such organisms compared to mammals in order to identify potential target processes for antimicrobial drug development.
Mass Spectrometry of Glycerolipids. The simplest glycerolipids contain only fatty acid substituents esterified to the glycerol backbone, and we have developed positive ion ESI/MS?MS methods for charactering both neutral acyl glycerols nad fatty acids as LI+ adducts, which yield a much more informative fragmentation pattern than the protonated species or adducts with other metal ions.
Glycerophospholipids contain fatty acids esterified to the sn-1, sn-2, or both of those carbon atoms and a phosphodiester moiety linked to a polar head-group in the sn-3 position, such as choline, ethanolamine, inositol, serine, or glycerol.
Glycerophosphocholine lipids are most readily characterized by ESI/MSn as Li+ adducts in positive ion mode, which yield more informative tandem spectra than do species that are protonated or adducted with other examined ions. GLycerolipids with ethanolamine, serine, inositol, or glycerol head-groups are in general more readily characterized by negative ion ESI/MSn by collisionally activated dissociation (CAD) of [M-H]- ions, although positive ion spectra of metal adducts of these compounds may also be informative in some cases. We have described approaches for sturctural characterization of all major clycerolipid classes by ESI/MSn and have prepared a recent of these studies. We have also recently published a description of computer algorithm for automatic identification of glycerophospholipid molecular species from raw electronic mass spectrometric data.
Mass Spectrometry of Sphingolipids. Ceramide is a relatively simple sphingolipid that contains a fatty acid in an amide linkage to the amine moiety of the long-chain base. Ceramide molecular species differ in the identity of the fatty acid substituent and of the long-chain base and can be characterized a Li+ adducts by positive ion ESI/MSn or by negative ion analysis.
We have also developed ESI/MSn approaches for structural characterization of the sphingolipids sphingomyelin, sulfatide, and glycosphingolipids, all of which are substituted ceramides. In sphingomyelin phosphocholine is linked via a phosphodiester group to the hydroxyl moiety on the long-chain base of serine, and in glycosphingolipids, various carbohydrates are linked to that hydroxyl moiety. Sulfatide is a glycosphingolipid in which the carbohydrate substituent is galactose substituted with a sulfate group. We have also prepared recent reviews describing our approaches to the structural characterization of sphingolipid molecular species.
Mass Spectrometry of Complex Lipids from Pathogenic Microorganisms. We have applied the ESI/MSn methods that we have developed to the characterization of complex lipids in pathogenic microorganisms including Leishamania major, the etiologic agent in visceral and cutaneous Leishmaniasis; pathogenic bacteria, including the gram-negative organisms Salmonella typhimurium and Escherichia coli and the gram-positive organism Streptococcus pyogenes; adn mycobacterial species, including Mycobacterium tuberculosis.
Visceral Leishmaniasis is a fatal illness and cutaneous Leishmaniasis frequently afflicts U.S. soldiers in Iraq as the so-called “Baghddad boil.” We have identified a novel pathway for sphingolipid metabolism involved in ethanolamine biosynthesis in these organisms that is not used in mammals, and it therefore represents a potential target for development of therapeutic drugs with antimicrobial activity towardd Leishmania major.
Among gram-negative bacteria, Salmonella species are frequent causes of gastrointestinal illnesses, and E. coli is the organism that most commonly causes urinary tract infections in humans. We have characterized mechanisms underlying resistance to antimicrobial agents in Salmonella that involve modifications of complex lipids in the cell envelope. Our findings could facilitate the design of more effective antibiotics against gram-negative bacteria, such as Salmonella and E. coli.
We have also characterized a novel enzyme involved in the synthesis of phosphatidylinositol mannosides and have developed MS methods to structurally characterize such molecules in the cell wall of Mycobacterium tuberculosis, the causative organism in human tuberculosis, that might also represent a target for development of antimycobacterial drugs, which is an area of intense current interest in view of the emergence of multi-drug resistant strains of this organism.
Reviews. Our MS approaches to characterization of glycerolipids and sphingolipids have recently been reviewed in the following book chapters:
- Hsu FF & Turk J. Mechanisms for phospholipid fragmentation during electrospray ionization mass spectrometry with collision-induced dissociation. In Modern Methods for Lipid Analysis by Liquid Chromatography-Mass Spectrometry and Related Techniques (WC Byrdwell, ed.), pp. 61-179, American Oil Chemists Society Press, Champaign, IL (2005).
- Hsu FF & Turk J. Tandem quadrupole mass spectrometry of sphingomyelin. In The Encyclopedia of Mass SPectrometry. Volume 3: Biological Applications, Part B. Carbohydrates, Nucleic Acids, and other Biological Compounds (Caprioli R & Gross ML, eds.), pp. 430-437, Elsevier Ltd., Oxford, UK (2006).
- Hsu FF& Turk J. Tandem quadrupole mass spectrometry of sulfatides. In The Encyclopedia of Mass Spectrometry, Volume 3: Biological Applications, Part B. Carbohydrates, Nucleic Acids, and other Biological Compounds (Caprioli R & Gross ML, eds.), pp. 473-492, Elsevier Ltd., Oxford, UK (2006).
The complete Lipid Mass Spectrometry: Core Technologic Research and Development document can be downloaded below:
Lipidomics Research Poster (47-page pdf)