research articles Szapacs et al. Table 2. Carboxylated Electrophile Adducts Found in ApoA1 from Plasma Supplemented with PLPBSO and Subjected to Oxidation with AIPH sequences modiﬁed QLNLK*LLDNWDSVTSTFSK QKLH*ELQEK AH*VDALRTHLAPYSDELR TH*LAPYSDELR LEALK*ENGGAR LEALK*ENGGAR LAEYHAK*ATEHLSTLSEK positions K45 H135 H155 H162 K182 K182 K195 mass shift 210 128 228 226 158/142 128 156 vidin and imaged using the LiCOR Odyssey imaging system (Licor, Lincoln, NE). Conﬁrmation of ApoA1 Adduction by Immunoblotting. Both oxidized and unoxidized plasma were rotated with 100 µ L of Protein A-Agarose slurry (see above) to remove IgG light chain, which nearly coelutes with ApoA1. The remaining plasma proteins were resolved on a 10% NuPAGE Novex Bis- Tris gel using MES running buffer and the proteins were electrophoretically transferred to a PVDF membrane. The blocked membrane was incubated with rabbit polyclonal antibody raised to the RLAEYHAKATEH peptide of ApoA1 and immunoblot images were acquired with the LiCOR Odyssey imaging system. Identiﬁcation of Biotinylated Lipid Oxidation Induced Adducts on ApoA1. PLPBSO supplemented oxidized plasma was incubated with 100 µ L of streptavidin sepharose slurry followed by the stringent washing as described above. The immobilized biotinylated proteins then were eluted from the beads by hydrolysis of the phospholipid fatty acyl ester bonds with 15% NH4OH for 12-15 h at room temperature. The eluted proteins were resolved on a 10% NuPAGE Novex Bis-Tris gel and stained with colloidal blue and the ApoA1 band (∼25-28 kDa) was excised and subjected to in-gel tryptic digestion.41 The eluted tryptic peptide solution was evaporated to dryness and the peptides were resuspended in 50 µ L of 0.1% formic acid in water for LC-MS-MS analysis. MS-MS spectra were acquired as described above, except that gas-phase fraction- ation was not used; precursor ions for MS-MS were selected from full scans over the range m/z 300-2000. The data were analyzed with the P-Mod algorithm42 using ApoA1 tryptic peptide sequences to detect MS-MS spectra corresponding to carboxylated electrophile(s) (adduct type(s)) KODA (imine adduct) 7-oxoheptanoic acid (imine adduct) HODA (Michael adduct nonreduced) KODA (Michael adduct, nonreduced or ketoamide adduct) 8-oxoocatanoic acid (carbinolamine and imine adduct forms) 7-oxooctanoic acid (imine adduct) 9-oxononanoic acid (imine adduct) mass-modiﬁed ApoA1 peptides. P-Mod is available for down- load at http://www.mc.vanderbilt.edu/msrc/bioinformatics/ software.php. Additional analyses used a similar algorithm in development in our laboratory, called MonsterMod, which is similar to P-Mod, but includes improvements in the scoring and statistical models for generation and evaluation of sequence- to-spectrum matches (Ham, A. L.; Wernke, G. R.; Tabb, D. L.; Kim, H. Y.; Liebler, D. C., manuscript in preparation). Identiﬁcation of HNE Adduction Sites in ApoA1. To char- acterize the formation of HNE adducts in HNE-treated samples, isolated HDL was treated with HNE at concentrations of 1 µ M, 10 µ M, 100 µ M, and 1 mM. The adducts were stabilized by reduction with 30 mM NaBH4. The HNE treated HDL proteins were resolved by SDS-PAGE on a 10% NuPAGE Novex Bis-Tris gel and stained using colloidal blue followed by tryptic in-gel digestion of the ApoA1 band between ∼20-28 kDa. To deter- mine HNE adducts formed endogenously during HDL oxida- tion, HDL was incubated with AIPH initiator overnight at concentrations of 0, 100, 1000, and 5000 µ M followed by NaBH4 reduction. The HDL proteins then were processed and analyzed as described immediately above. In both cases, the recovered peptides were analyzed by LC-MS-MS and the data then were searched with the P-Mod. Mass shifts of 158 corresponded to NaBH4-reduced Michael adducts. Results Strategy for Identiﬁcation of Proteins Adducted by PLPBSO and Mapping of Adducts. The use of PLPBSO as a probe to identify protein targets of phospholipid electrophiles is depicted in Figure 1. Oxidation of PLPBSO with AIPH yields Figure 4. Isolated HDL treated with HNE and oxidized with AIPH in different concentrations. They are visualized with anti-HNE (left) or colloidal blue (right). Note the migration shift seen in the colloidal blue stained gel in the ApoA1 band between 20-26 kDa is induced by oxidation, but not by HNE. 4240 Journal of Proteome Research Vol. 7, No. 10, 2008