Affinity Capture and MALDI-MS of Cytokines from Mouse Serum Gregory B. Hurst 1 , Stephen J. Kennel 2 , Linda J. Foote 2 , and Michelle V. Buchanan 1,2 Chemical and Analytical Sciences Division 1 and Life Sciences Divison 2 Oak Ridge National Laboratory, Oak Ridge TN 37831 Inflammation is a condition associated with a wide variety of diseases. 1 The inflammatory response involves changes in serum levels of some cytokines. We are working toward a method for screening cytokine levels in mutant mouse populations as a possible means for earlier detection of genotypes leading to chronic inflammatory diseases. We describe a method for purifying targeted cytokines from serum and cell culture supernatants using affinity capture followed by MALDI-MS analysis. 2 Experimental: Antibodies to various cytokines were attached to 1 μ m diameter aminopolystyrene beads. 3 The beads were incubated with cell culture supernatants, or a solution of a recombinant version of the cytokine(s) of interest in 5 mg/mL BSA in PBS. The beads were recovered by centrifugation and washed. Captured cytokines were eluted from the beads by adding MALDI matrix solution, either sinapinic acid (SA) or α -cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile/50% aqueous 0.1% trifluoroacetic acid. The supernatant was transferred to a MALDI sample plate pre-treated with a thin layer of the appropriate matrix deposited from a volatile solvent. 4,5 MALDI-TOF measurements were performed using either a Voyager DE or Voyager Elite (PerSeptive Biosystems). Multiple spectra were obtained from each sample spot and those with highest S/N were subsequently averaged. Results: Figure 1 shows MALDI spectra of several recombinant cytokines (R&D Systems), each at the 100 ng level. The spectra were obtained using SA matrix, except interleukin-6 (IL- 6), for which better sensitivity was obtained using CHCA. Multiple peaks in the interleukin-2 (IL-2) spectrum are consistent with the presence of both N-terminal methionyl and native versions of the protein in the commercial product. The reason is not clear for large difference between the measured and calculated m/z for interferon- γ (IFN-γ29 . A multiplex-capture experiment was performed (data not shown) in which a mixture of affinity beads, each derivatized with an antibody to tumor necrosis factor- α (TNF-α29 , IL-2, or IFN-γ, was challenged with a mixture of these three cytokines. All three cytokines were detected at the 20 ng level. However, spectral overlap was observed between TNF- α and the largest IL-2 species, which differ in mass by only 17 Da. Multiplexing is desirable because cytokines can act in concert in biological systems. 1 Figure 2 shows results from affinity capture of native TNF- α from supernatants of a series of lipopolysaccharide (LPS)-treated mouse cell (macrophage or spleen) cultures. ELISA measurements of TNF- α were performed before and after capture, as shown in the figure. The qualitative trend is that a larger TNF- α signal is observed in the MALDI spectrum from those samples containing higher amounts of TNF- α by ELISA. Some non-specific capture was observed, but it did not interfere with detection of the TNF- α; we observed similar results from spiking recombinant TNF- α into mouse serum, followed by affinity capture and MALDI analysis. 3 Higher-mass forms (including glycoforms) 6 of TNF-α were not observed in this