DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

34. Affinity Capture and Mass Spectrometry of Targeted Proteins in Mice

Stephen J. Kennel¹, Gregory B. Hurst², Linda J. Foote¹, and Michelle V. Buchanan^1,2

¹Life Sciences Division and ²Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6124

buchananmv@ornl.gov

We are developing new mass spectrometry-based methods for large-scale screening of targeted proteins in mice, taking advantage of the ability of mass spectrometry to detect compounds sensitively and to identify both normal and modified proteins unambiguously. In this initial study, we are working with the Mammalian Genetics and Development Section at ORNL to develop a method for large-scale screening of cytokine levels in mouse serum samples as a means to detect subtle abnormalities leading to chronic inflammatory diseases. Modified levels of cytokines in serum are indicative of inflammation, a condition associated with a wide variety of disease states. The new methodology involves capture of targeted cytokines from mouse serum onto antibody-derivatized aminopolystyrene beads, washing, elution, and analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The selectivity of the affinity separation is complemented by the m/z measurement capability of mass spectrometry, providing speed and specificity advantages over conventional ELISA techniques. Tumor necrosis factor a (TNF-a), a 17 kDa proinflammatory cytokine that has a relatively high serum concentration in acute reactions and seems to be an early effector in inflammatory cytokine cascades, has been used as a model analyte (Hurst, G.B.; Kennel, S.J.; Foote L.J.; Buchanan, M.V. Anal. Chem. 1999, 71, 4727-4733). In parallel with MALDI-MS, experiments using 125I-labeled TNF-a and gamma detection allow independent optimization of the affinity capture, and indicate that the capture methodology is viable from <100 pg/mL to >50 ng/mL. MALDI-MS currently allows reliable detection down to 1 ng TNF-a in an initial 100 mL sample volume (mouse limited), and we are working to improve this figure. To demonstrate selectivity, TNF-a spiked into mouse serum can be concentrated onto the beads and detected by MALDI-MS with little interference from the many other components present in serum. Control experiments indicate that non-specific binding is minor. Preliminary MALDI-MS results on other cytokines (IL-6, IL-1b, IL-2, and IFN-g) indicate that the MALDI matrix conditions must be carefully optimized for each cytokine to allow sensitive detection.

Research sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by Lockheed Martin Energy Research Corp. for the U. S. Department of Energy under Contract No. DE-AC05-96OR22464.