The online, selective isolation of protein-ligand complexes using cobalt(II)-coated paramagnetic affinity beads (PABs) and subsequent liquid chromatography-mass spectrometry (LC-MS) determination of specifically bound ligands is described. After in-solution incubation of an analyte mixture with His-tagged target proteins, protein-analyte complexes are mixed with the Co(II)-PABs and subsequently injected into an in-house built magnetic trapping device. Bioactive ligands bound to the protein-Co(II)-PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are online eluted toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily lowering the magnetic field. The proof-of-principle is demonstrated by using commercially available Co(II)-PABs in combination with the His-tagged human estrogen-receptor ligand-binding domain. The system is characterized with a number of estrogenic ligands and nonbinding pharmaceutical compounds. The affinities of the test compounds varied from the high micromolar to the subnanomolar range. Typical detection limits are in the range from 20 to 80 nmol/L. The system is able to identify binders in mixtures of compounds, with an analysis time of 9.5 min per mixture. The standard deviation over 24 h is 9%.

This article is available online at http://www.jlr.org Supplementary key words drug therapy/hypolipidemic drugs • adipocytes • fatty acid/biosynthesis • omega-3 fatty acids • cytochrome P450 • polyunsaturated fatty acid Niacin (vitamin B3) treatment reduces CVD and atherosclerosis development ( 1 ). These benefi cial effects are mediated, in part, by lowering circulating levels of LDLcholesterol, VLDL-TG, and lipoprotein(a) ( 2 ), as well as by increasing HDL-cholesterol ( 3 ). In addition, prolonged niacin treatment also decreases plasma, adipose tissue, and vascular infl ammation ( 4, 5 ), which might contribute to reducing CVD. The induction of these benefi cial effects after prolonged niacin treatment is in striking contrast to the unwanted acute niacin effects.Acutely, niacin binds to the inhibitory hydroxycarboxylic acid receptor 2 (HCA 2 ) (previously known as GPR109A). In adipocytes, this leads to an inhibition of adipocyte lipolysis followed by an acute reduction of plasma nonesterifi ed fatty acid (NEFA) levels. Lowering NEFA levels causes metabolic stress ( 6, 7 ), which increases stress hormone levels ( 8-12 ) after niacin treatment. In the skin Langerhans cells and keratinocytes, acute niacin binding to the

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