Affinity Capture and MALDI-MS of Cytokines from Mouse Serum

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 mm 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 a-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 fro