FIGURE 2.

Quantification of regiospecific chlorination of the Tyr residue in lipid-free and HDL-associated apoA-I exposed to HOCl or the MPO-H₂O₂-NaCl system. Lipid-free apoA-I (10 μm) (A) or HDL₃ (0.5 mg/ml, ∼12.5 μm apoA-I) (B) was exposed to 100 or 180 μm HOCl, respectively (striped bars) or to 100 or 180 μm H₂O₂, respectively, in the MPO-H₂O₂-chloride system (solid bars) for 60 min at 37 °C in phosphate buffer (20 mm sodium phosphate, 100 μm DTPA, pH 7.4). The MPO system contained 100 nm enzyme and 100 mm sodium chloride. The reaction was terminated with l-methionine. A tryptic digest of apoA-I or HDL₃ was analyzed by selected reaction monitoring mass spectrometry analysis. Four transitions were selected for each Tyr-containing precursor and product peptide to quantify the product yield of chlorinated tyrosine residues. The product yield of 3-chlorotyrosine was calculated as described under “Experimental Procedures.” Results are the means ± S.D. of three independent experiments, with triplicate determinations per experiment.