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B.A., B.S., University of Miami
Ph.D., University of Massachusetts at Amherst
My research group is primarily involved in developing new methods for measuring lipid species-using comprehensive, automated lipid profiling (lipidomics, if you will) and using ultratrace methods based on laser-induced fluorescence. We use these methods to measure primary fatty acid amide (PFAM) distribution in mammalian systems and to understand the metabolism of PFAM's. Following are brief summaries of various projects.
Fatty acid amides (PFAM's) comprise a family of neutral lipids that is related to other classes of N-acyl amines, such as N-acyl amino acids, N-acylethanolamines, and more complicated species like sphingomyelins and ceramides. Our group has developed a number of analytical methods for quantifying amides, most of them based on liquid chromatography (HPLC and TLC) and gas chromatography (GC) with fluorescence and mass spectrometric detection.
Mass spectrometry is the workhorse of the 'omics disciplines, but a critical part of making quantitative mass spectral measurements is sample preparation. Research in our group involves developing high efficiency, multidimensional chromatographic methods for isolating specific classes of neutral lipids, such as the fatty acid amides, and for automating the sample preparation of neutral lipids for mass spectrometry.
Sample size and final instrument sensitivity dictates the scale of the sample preparation procedure. For large samples (whole murine organs, for example), we use TLC and normal phase solid phase extraction (1, 12). For smaller samples (cultured cells) we use HPLC. Ultimately, we will use microfluidic platforms for very small samples (how small? Single cells? Organelles?). GC/MS and LC/APCI/MS or LC/ESI/MS provide adequate sensitivity for the larger scales (tens of fmol), but the smallest scale will require SIM/SIM MS/MS (amol). For complex lipids, MS/MS is also necessary for identifying the structural components of the individual lipids, as there are many isobars resulting from positional and geometric isomers.
As impressive as MS/MS can be, at its most sensitive, laser-induced fluorescence (LIF) is still better by at least 3 orders of magnitude-capable of sub-pM concentration detection limits and sub-zmol mass detection limits (3). To put that in perspective, one nL at pM concentration is 1 zmol of material, or about 600 molecules. A 50 µm diameter cell is roughly 65 pL in volume, so any molecule in the cell at a concentration of 1 µM will comprise only 65 amol of material. Loss of sample during the sampling process and dilution from dead volume in the sampling device can kill the ability to measure trace species. LIF can help.if you can tag your molecule or otherwise use some kind of molecular beacon to show when it has been expressed (this works for genomics and proteomics particularly well). We have been using unique ways to tag amines, acids, and amides with fluorophores, and we then separate those species using capillary electrophoresis, microcolumn liquid chromatography, and capillary electrochromatography. Detection is with custom high efficiency detection systems (3).
1. Sultana, T.; Johnson, M. E. 'Sample Preparation and Gas Chromatography of Primary Fatty Acid Amides' J. Chromatogr. B , 2006 , 1101 , 278-285
2. Johnson, M. E.; Carpenter, T. S. 'The Use of Solid Phase Supports for Derivatization in Chromatography and Spectroscopy' Applied Spectroscopy Reviews , 2005 , 40 , 1-22
3. Johnson, M. E.; Landers, J. P. ' Fundamentals and Practice for Ultrasensitive Laser-Induced Fluorescence Detection in Micro-Analytical Systems' Electrophoresis 2004 , 25 , 3513-3527
4. Merkler, D. J.; Chew, G. H.; Gee, A. J.; Merkler, K. A.; Sorondo, J-P. O.; Johnson, M. E. 'Oleic Acid Derived Metabolites in Mouse Neuroblastoma N 18 TG 2 Cells' Biochemistry 2004 , 43 , 12667-12674
5. Carpenter, T.; Poore, D.; Gee, A. J.; Deshpande, P.; Merkler, D. J.; Johnson, M. E. 'Liquid Chromatographic Separation of Fatty Acid Amides and N-Acyl Glycines for Biological Assays' J. Chromatogr. B: Biomed. Appl. 2004 , 809 , 15-21
6. Stokes, J. C.; Johnson, M.E. 'Resolution in Sub-Micrometer Particle Separations by Capillary Electrophoresis' Microchem. J. 2004 , 76 , 121-129
7. Gallaher, D. L.; Johnson, M. E. 'Nonaqueous Capillary Electrophoresis of Fatty Acids Derivatized with a Near-infrared Fluorophore' Anal. Chem. 2000 , 72(9), 2080-2086
8. Gee, A. J.; Groen, L.; Johnson, M. E. 'Ion Trap Mass Spectrometry of Trimethylsilylamides Following Gas Chromatography' J. Mass Spectrom. 2000 , 35(3) , 305-310
9. Gallaher, D. L.; Johnson, M. E. 'Development Of Near-Infrared Fluorophoric Labels For The Determination Of Fatty Acids Separated By Capillary Electrophoresis With Diode Laser Induced Fluorescence Detection' Analyst 1999 , 124 , 1541-1546
10. Gee, A. J.; Groen, L.; Johnson, M. E. 'Determination of Fatty Acid Amides as Trimethylsilyl Derivatives by Gas Chromatography with Mass Spectrometric Detection' J. Chromatogr. A 1999 , 849 , 541-552
11. Feng, L.; Johnson, M. E. 'Selective Fluorescence Derivatization and Capillary Electrophoretic Separation of Amidated Amino Acids' J. Chromatogr. A 1999 , 832 , 211-224
and theses/dissertations (view the more recent ones electronically here )
12. Sultana, T. 'Primary fatty acid amides in mammalian tissues: Isolation and analysis by HPTLC and SPE in conjunction with GC/MS' Ph.D., Duquesne University, April, 2005
13. Carpenter, T. S. 'Derivatization at Ultratrace Levels on Solid Phase Sorbents' Ph.D., Duquesne University, April, 2005
14. Gee, A. J. 'Strategies for Detection and Quantitation of Biologically Important Compounds at Trace and Ultra-Trace Levels' Ph.D., Duquesne University, August, 2000
15. Gallaher, D. L. 'Strategies for Near-Infrared Diode Laser-Induced Fluorescence Detection of Bioactive Analytes in Capillary Electrophoresis' Ph. D., Duquesne University, October, 1999
Funding
NIH National Institute for Neurological Disorders and Stroke (NS 038443)
Office Phone: 412.396.5278
Email: johnsonm@duq.edu |
