Lipid Electrophile Adducts of ApoA1 research articles Figure 1. Structure of PLPBSO and application to identiﬁcation of protein targets of phospholipid electrophiles. See text for discussion. nologies, LLC, Phoenix, AZ) packed with 5 µ m, 300 Å Jupiter C18 (Phenomenex, Torrance, CA). Liquid chromatography was carried out at ambient temperature at a ﬂow rate of 0.2 mL min-1 using a gradient mixture of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile. Peptides eluting from the capillary tip were introduced into the LTQ nanoelectrospray source with a capillary voltage of approxi- mately 2 kV. A gas-phase fractionation method39 was used to increase identiﬁcations of lower abundance proteins that were present. Each sample was run three times, ﬁrst with a full scan and precursor selection in the range of m/z 400-600, then m/z 600-900, and ﬁnally m/z 900-2000. Five data-dependent MS-MS scans were performed on the selected precursor ions from each full scan. MS-MS spectra were recorded using dynamic exclusion of previously analyzed precursors for 30 s with a repeat of 1 and a repeat duration of 2. MS-MS data were evaluated using the Sequest algorithm40 and the IPI Human database, which included both forward and reverse sequences for all entries. Sequest outputs were organized and analyzed with a custom database utility called CHIPS (Complete Hierarchical Integration of Protein Searches). CHIPS allows the user to ﬁlter Sequest outputs based on Sequest output param- eters such as XCorr, Rsp, and Sp scores. In this case, both the native oxidized plasma (control) and PLPBSO supplemented oxidized plasma MS-MS data were ﬁltered using Xcorr score thresholds of g2 for 1+ peptides, Xcorr g2.5 for 2+ peptides, and Xcorr g3 for 3+ peptides with Rsp e 5 and Sp g 350. The false positive rate for peptide identiﬁcation based on reversed sequence identiﬁcations with these criteria was 13.4%. The requirement that protein identiﬁcations require at least two distinct peptides per protein reduced the false positive rate for protein identiﬁcation to 0.4%. Proteins that were identiﬁed after biotin-streptavidin capture in both the control and PLPBSO supplemented oxidized plasma sample were removed and the remaining identiﬁed proteins were considered covalently ad- ducted by PLPBSO. Figure 2. Base hydrolysis of biotinylated proteins in various concentrations of NH4OH and different temperatures shown in Alexa Fluor 680-conjugated streptavidin Western blot (left) and visible coomassie-stained gel (right). Disappearance of biotin in the Western shows the degree of hydrolysis of the phospholipid headgroup. The visible gel indicates the stability of the proteins under the hydrolysis conditions. Table 1. Sequence Coverage of Major Proteins Captured by PLPBSO Following Oxidation in Human Plasma protein albumin alpha-1-antitrypsin apolipoprotein A1 apolipoprotein A2 apolipoprotein A4 apolipoprotein E complement C3 sequence coveragea 58.6% 26.9% 68.3% 49.0% 34.4% 30.6% 24.8% a Sequence coverage is deﬁned as the fraction of the protein sequence accounted for by MS-MS-identiﬁed peptides. Figure 3. Immunoblot conﬁrmation of PLPBSO adducts on ApoA1. Oxidation induces a migration shift of ApoA1 in SDS- PAGE analysis and streptavidin labels the PLPBSO-modiﬁed ApoA1 protein. Oxidized plasma was immunoblotted with poly- clonal ApoA1 antibody followed by Alexa ﬂuor 680 antirabbit (left) or with antistreptavidin (right). Conﬁrmation of Biotinylated Lipid Adducts Using Immu- noblotting Techniques. Proteins captured on streptavidin beads as described above were eluted using 1 M formic acid in acetonitrile/water (3:2, v/v) overnight at room temperature after washing with the solutions described above. The super- natant was removed and the beads were rinsed with 2 × 100 µ L of 50 mM ammonium bicarbonate. The combined elution mixture was evaporated under vacuum. The eluted proteins were resolved by 10% NuPAGE Novex Bis-Tris gel and the resolved proteins then were transferred to a PVDF membrane, which was incubated with Alexa Fluor 680-conjugated strepta- Journal of Proteome Research Vol. 7, No. 10, 2008 4239