Phospholipid−Protein Adducts of Lipid Peroxidation - American

Chem. Res. Toxicol. 2007, 20, 227-234

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Phospholipid-Protein Adducts of Lipid Peroxidation: Synthesis and Study of New Biotinylated Phosphatidylcholines Keri A. Tallman,† Hye-Young H. Kim,† Jian-Xin Ji,† Matthew E. Szapacs,‡ Huiyong Yin,† T. J. McIntosh,| Daniel C. Liebler,‡,§ and Ned A. Porter*,† Department of Chemistry, Department of Biochemistry, and Mass Spectrometry Research Center, Vanderbilt UniVersity, NashVille, Tennessee 37235, and Department of Cell Biology, Duke UniVersity Medical Center, Durham, North Carolina 27712 ReceiVed NoVember 21, 2006

Oxidative stress gives rise to a number of electrophilic aldehydes from membrane phospholipids, and these compounds have been linked to pathophysiologic events associated with the progression of cardiovascular disease. A headgroup biotinylated phosphatidylcholine (PC) has been prepared, and its oxidation chemistry has been studied. Biotin or biotin-sulfoxide groups were attached to PC at the ammonium headgroup via a di-ethylene glycol link. The modified phospholipids have calorimetric and colloidal properties similar to those of the parent. The oxidation of PLPBSO (the biotin-sulfoxide analogue of 1-palmitoyl-2-linoleoylglycerylphosphatidylcholine, PLPC) was studied under a variety of conditions. PLPBSO, like PLPC, undergoes oxidation to give electrophiles that adduct to small model peptides as well as to isolated proteins such as human serum albumin. PLPBSO incorporates into human blood plasma, and treatment of the plasma with water soluble free radical initiators gives rise to a number of biotinylated plasma proteins that can be isolated via (strept)avidin affinity. Isolated peptide or proteinlipid adducts can be identified by proteomics analyses, and studies on model peptides show that phospholipid-protein adduction sites can be identified by known algorithms. Biotinylated lipids such as PLPBSO and modern proteomics tools would appear to provide a new approach to exploring the chemistry and biology of membrane peroxidation associated with oxidative stress. Introduction Oxidative stress generally leads to the reaction of polyunsaturated lipids with oxygen, and this process, lipid peroxidation, gives rise to a host of products (1, 2). A number of diseases in which environmental and lifestyle factors play a role have been linked to oxidative stress. These include atherosclerotic cardiovascular disease (3, 4), neurodegenerative disorders (5), and cancer (6-8), among others. Although hydroperoxides and peroxides are the primary products of lipid peroxidation, decomposition of these unstable intermediates gives rise to a number of electrophilic aldehydes with significant bioactivity (9). Among the most prominent and well-studied of these electrophiles are the cytotoxic aldehydes 4-hydroxy-nonenal (4-HNE) (10), 4-hydroperoxy-nonenal (4HPNE), 4-oxo-nonenal, and 4-hydroxy-hexenal (11-13). These reactive products can deplete cellular GSH (14) through adduct formation, leading to apoptosis. They also have the potential to interfere with cell cycle events and manipulate protein expression. The peroxidation of membrane phospholipids, such as those present in circulating lipoproteins, leads to electrophilic products that retain short-chain aldehydes esterified to the phosphatidylcholine head group. Products of this type have been shown to promote the entry of monocytes into the vessel wall (15). In the progression of atherosclerosis, they can also serve as potent * To whom correspondence should be addressed. Tel: 615-343-2693. Fax: 615-343-5478. E-mail: [email protected]. † Department of Chemistry, Vanderbilt University. ‡ Department of Biochemistry, Vanderbilt University. § Mass Spectrometry Research Center, Vanderbilt University. | Duke University Medical Center.

ligands for the macrophage scavenger receptor CD36, actively promoting the uptake of oxidized lipids, and ultimately leading to the transformation of macrophages into foam cells (16). Therefore, understanding the reactivity profiles of the electrophilic products of lipid peroxidation, particularly those derived from phospholipids, may provide insight into the pathophysiology of oxidative stress. We report here on the synthesis, characterization and study of glycerylphosphatidycholine (PC) analogues, DPPB and PLPB, that are modified in the headgroup with a biotin functional group, along with the corresponding biotin-sulfoxide derivative PLPBSO. The biotinylated phospholipids, which retain the zwitterionic headgroup of the lipid class and have physical properties similar to those of analogous phosphatidylcholines, undergo lipid peroxidation to give products that covalently modify proteins.

Our studies suggest that such biotinylated phospholipids may prove to be generally useful in the identification of protein targets of phospholipid-derived electrophiles and, therefore, provide a means to identify important biomarkers of oxidative stress.

10.1021/tx600331s CCC: $37.00 © 2007 American Chemical Society Published on Web 01/12/2007

228 Chem. Res. Toxicol., Vol. 20, No. 2, 2007

Tallman et al.

Experimental Procedures

with H2O. The sticky precipitate was dissolved in acetone and concentrated. After extensive drying, the product was isolated as an off-white foam (6.7 g, 76%). 1H NMR (acetone-d6) δ 7.34 (m, 8H), 7.17 (br t, J ) 5.4 Hz, 1H), 6.95 (t, J ) 7.2 Hz, 8H), 6.80 (t, J ) 7.2 Hz, 4H), 6.32 (br s, 1H), 6.13 (br s, 1H), 4.78 (t, J ) 4.8 Hz, 1H), 4.49 (dd, J ) 5.1, 7.8 Hz, 1H), 4.31 (m, 1H), 4.08 (m, 2H), 3.99 (m, 2H), 3.72 (m, 2H), 3.63 (m, 6H), 3.51 (t, J ) 5.4 H, 2H), 3.39 (m, 2H), 3.30 (s, 6H), 3.22 (m, 1H), 2.92 (dd, J ) 5.1, 12.6 Hz, 1H), 2.69 (d, J ) 12.6 Hz, 1H), 2.19 (t, J ) 6.9 Hz, 2H), 1.75 (m, 2H), 1.61 (m, 2H), 1.43 (m, 2H); HRMS (ES+) calculated (M - BPh4), 447.2641; observed, 447.2640. Synthesis of PLPB. 2,4,6-Triisopropylbenzenesulfonyl chloride (0.22 g, 0.73 mmol) was added to a solution of 1-palmitoyl-2linoleoylglycerylphosphtidylcholine (0.40 g, 0.59 mmol) in pyridine (12 mL) at 40 °C. After 30 min, 6 (0.63 g, 0.82 mmol) was added and the reaction mixture stirred for 4 h. The reaction was quenched with MeOH and concentrated. Purification by column chromatography (CH2Cl2/MeOH/H2O, 68:30:2 to 65:30:5) afforded the product as a pale-yellow powder (0.10 g, 16%). 1H NMR (MeOH-d4) δ 5.34 (m, 4H), 5.25 (m, 1H), 4.51 (dd, J ) 4.8, 7.8 Hz, 1H), 4.44 (dd, J ) 2.7, 12.0 Hz, 1H), 4.32 (m, 3H), 4.17 (dd, J ) 6.9, 12.0 Hz, 1H), 4.02 (t, J ) 6.0 Hz, 2H), 3.98 (m, 2H), 3.79 (m, 2H), 3.71 (m, 2H), 3.65 (m, 4H), 3.54 (t, J ) 5.4 Hz, 2H), 3.36 (t, J ) 5.4 Hz, 2H), 3.27 (s, 6H), 3.24 (m, 1H), 2.94 (dd, J ) 4.8, 12.6 Hz, 1H), 2.77 (t, J ) 6.0 Hz, 2H), 2.73 (d, J ) 12.9 Hz, 1H), 2.34 (t, J ) 7.5 Hz, 2H), 2.31 (t, J ) 7.5 Hz, 2H), 2.24 (t, J ) 6.9 Hz, 2H), 2.06 (q, J ) 6.0 Hz, 4H), 1.68 (m, 8H), 1.44 (m, 2H), 1.29 (m, 38H), 0.89 (m, 6H); 13C NMR (MeOH-d4) δ 176.2, 174.7, 174.4, 130.9, 130.8, 129.1, 129.0, 71.8, 71.7, 71.4, 71.1, 66.0 (m), 65.8, 64.9 (m), 63.7, 63.3, 61.6, 60.6 (m), 57.0, 53.3, 41.1, 40.4 (m), 36.7, 35.1, 34.9, 34.9, 34.7, 33.1, 32.7, 30.8, 30.7, 30.5, 30.4, 30.31, 30.26, 30.2, 29.7, 29.5, 28.2, 26.9, 26.6, 26.0, 23.8, 23.7, 14.6; HRMS (MALDI) calculated (M + H), 1101.7266; observed, 1101.7206. Synthesis of PLPBSO. A solution of NaIO4 (18) (22 mg, 0.10 mmol) in H2O (0.5 mL) was added to a solution of 15 (110 mg, 0.10 mmol) in MeOH (0.5 mL) at 0 °C. After 6 h, the product was purified by C18 SPE (0.5 g capacity). The cartridge was conditioned with MeOH (3 mL) and H2O (3 mL) and then the crude reaction mixture loaded. The cartridge was washed with H2O (3 mL) to elute the salts and the product eluted with MeOH. The product was isolated as a white powder (66 mg, 59%). 1H NMR (MeOHd4) δ 5.34 (m, 4H), 5.25 (m, 1H), 4.68 (m, 1H), 4.58 (dd, J ) 5.1, 8.7 Hz, 1H), 4.43 (dd, J ) 3.0, 12.0 Hz, 1H), 4.29 (m, 2H), 4.17 (dd, J ) 6.9, 12.0 Hz, 1H), 4.00 (t, J ) 6.3 Hz, 2H), 3.95 (m, 2H), 3.67 (m, 8H), 3.54 (t, J ) 5.7 Hz, 2H), 3.50 (m, 1H), 3.36 (t, J ) 5.4 Hz, 2H), 3.24 (s, 6H), 3.11 (m, 2H), 2.77 (t, J ) 5.7 Hz, 2H), 2.34 (t, J ) 7.8 Hz, 2H), 2.31 (t, J ) 7.5 Hz, 2H), 2.25 (t, J ) 7.2 Hz, 2H), 2.06 (q, J ) 6.6 Hz, 4H), 1.88 (m, 2H), 1.69 (m, 2H), 1.57 (m, 6H), 1.31 (m, 38H), 0.89 (m, 6H); 13C NMR (MeOH-d4) δ 176.0, 174.8, 174.4, 130.9, 130.8, 129.1, 129.0, 71.9, 71.8, 71.7, 71.3, 71.1, 70.6, 66.3 (d), 65.7 (d), 64.9 (d), 63.7, 60.4 (d), 59.4, 58.2, 55.2, 53.3, 40.4, 36.5, 35.1, 34.9, 33.1, 32.7, 30.8, 30.7, 30.5, 30.4, 30.3, 30.25, 30.19, 28.2, 28.1, 26.7, 26.6, 26.4, 26.0, 23.8, 23.7, 14.6; HRMS (MALDI) calculated (M + H), 1117.7215; observed, 1117.7276. Synthesis of DPPB. DPPB was synthesized by coupling of 1,2dipalmitoyl-sn-glycero-3-phosphate with 6 following the procedure described for PLPB. 1H NMR (CDCl3) δ 5.14 (m, 1H), 4.45 (m, 1H), 4.36-4.27 (m, 4H), 4.07 (dd, J ) 7.8, 12 Hz, 1H), 3.95 (m, 2H), 3.87 (m, 2H), 3.74 (m, 4H), 3.61-3.56 (m, 8H), 3.29 (br s, 6H), 3.09 (m, 1H), 2.79 (m, 2H), 2.24 (m, 6H), 1.65 (m, 2H), 1.53 (m, 4H), 1.21 (m, 52H), 0.83 (t, J ) 6.6 Hz, 6H); MS (ES+) calculated (M + H), 1077.73; observed, 1077.91. Modification of AcAVAGKAGAR by PLPBSO. AcAVAGKAGAR (1 mg/mL) and POPC/PLPBSO ) 5:1 were mixed and sonicated for 10 min to form the liposome. Then the reaction mixture was oxidized at 37 °C for 5 h, initiated by water soluble azo initiator AIPH (final concentration, 1 mM). It was reduced with NaCNBH3 (final concentration, 50 mM) and then desalted using zip tips prior to LC-MS/MS analysis. The mobile phase

General Methods and Materials. 1H and 13C NMR spectra were collected on a 300 or 400 MHz NMR. All reactions were carried out under an atmosphere of argon. THF and CH2Cl2 were dried using a solvent purification system. Commercial anhydrous DMF and CH3CN were used as received, and pyridine was freshly distilled from CaH2. 2-Bromoethanol was distilled from CaSO4/ Na2CO3, 2,4,6-triisopropylbenzenesulfonyl chloride (TiPSCl) was recrystallized from 10% SOCl2/hexanes, and iPr2EtN was distilled from CaH2. Purification by column chromatography was carried out on silica gel, and TLC1 plates were visualized with phosphomolybdic acid, Ninhydrin, or KMNO4/H2SO4. Human serum albumin (Sigma), DTT (Sigma), IAM (Sigma), monomeric avidin kit (Pierce), Streptavidin (Amersham Bioscience), NaCNBH3 (Aldrich), TPepK (AcAVAGKAGAR, NewEngland Peptides), 10% NuPage Novex bis-Tris precast mini gel (Invitrogen), Zip Tip (C18, Millipore), Simply Blue (Bio Rad), Streptavidin Alexa fluro-680 (Molecular Probe), PVDF membrane (Invitrogen), Blocking buffer (Rockland), Jupiter C-18 (5 µm) resin (Phenomenex), 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, AIPH (Wako, Japan), and biotinylated rabbit polyclonal antibody to HSA (ab24207) (Abcam Inc., Cambridge, MA) were obtained as indicated. MS samples were analyzed on a Thermo LTQ linear ion trap instrument equipped with a Thermo microelectrospray source, and a Thermo Surveyor pump and autosampler (Thermo Electron Corporation, San Jose, CA). LC-MS-MS analyses were done by reversed-phase chromatography on an 11 cm fused silica capillary column (100 µm i.d.) packed with Jupiter C-18 (5 µm) with the flow set at 200 µL/min. MS-MS spectra were acquired using either a data-dependent scan (DDS) followed by MS2 and MS3 or selected ion monitoring (SIM). Most of the DDSs were allowed to scan MS2 for four of the most intense precursors and MS3 for two of the most intense precursors. The relative collision energies of DDS and SIM were set at a maximum of 30% and 20%, respectively. Capillary temperature was 150 °C. Protein modification was analyzed by PerSpective Biosystem Voyger_DE STR (v5.10) using sinapinic acid as a matrix. The membrane was imaged using the LI-COR Odyssey imaging system (LI-COR, Lincoln, NE). Plasma Isolation. Whole blood from fasting, healthy subjects was collected in a 440 mL ACD blood collection bag (Baxter) containing the following: 2 g of dextrose monohydrate: 1.66 g of sodium citrate dihydrate; 188 mg of anhydrous citric acid; 140 mg of monobasic sodium phosphate monohydrate; and 17.3 mg of adenine. Plasma was subsequently isolated from red blood cells by centrifuging the whole blood bag at 4200 rpm for 10 min at 22 °C. Synthesis of 5. 2-Bromoethanol (2.0 mL, 0.028 mol) was added to a solution of 4 (5.5 g, 0.014 mol) and basic Dowex (15.3 g, 1.08kg/1eq) in EtOH (70 mL) and then heated to reflux. After 1 h, the Dowex was filtered off and the reaction mixture concentrated. The product (6.5 g, 90%) was isolated as a thick oil, which solidified upon drying under high vacuum. 1H NMR (D2O spiked with MeOH-d4) δ 4.45 (dd, J ) 4.8, 7.8 Hz, 1H), 4.26 (dd, J ) 4.5, 7.8 Hz, 1H), 3.90 (m, 2H), 3.82 (m, 2H), 3.48 (m, 10H), 3.23 (t, J ) 5.1 Hz, 2H), 3.15 (m, 1H), 3.06 (s, 6H), 2.84 (dd, J ) 4.8, 12.9 Hz, 1H), 2.62 (d, J ) 12.9 Hz, 1H), 2.11 (t, J ) 7.2 Hz, 2H), 1.48 (m, 4H), 1.27 (m, 2H); 13C NMR (D2O spiked with MeOH-d4) δ 177.8, 166.2, 70.7, 70.3, 69.9, 67.3 (m), 65.2, 63.1, 61.2, 56.4, 53.2 (m), 40.7, 39.8, 36.4, 28.9, 28.7, 26.2; HRMS (ES+) calculated (M - Br), 447.2641; observed, 447.2647. Synthesis of 6. A solution of NaBPh4 (17) (3.8 g, 0.011 mol) in H2O (50 mL) was added to a solution of 5 (6.0 g, 0.011 mol) in H2O (50 mL). A white, sticky precipitate formed immediately. After 30 min, H2O was decanted and the precipitate washed several times 1 1Abbreviations: PUFA, polyunsaturated fatty acid; MS, mass spectrometry; TLC, thin-layer chromatography; LC, liquid chromatography; ESI, electrospray ionization; CID, collision-induced dissociation; SIM, selective ion monitoring; SRM, selective reaction monitoring; PLPC, 1-palmitoyl, 2-linoleoyl glycerylphosphatidyl-choline; AIPH, 2,2′-azobis[2-(2-imidazolin-2-yl)-propane] dihydrochloride.

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Scheme 1. Outline of the Synthesis of Biotin-Modified Phosphatidylcholines

a Reagents: (a) formalin, NaCNBH , MeOH; (b) TFA, anisole, CH Cl ; (c) i. biotin, CDI, DMF; ii. 3; (d) 2-bromoethanol, Dowex, EtOH, reflux; (e) 3 2 2 NaBPh4, H2O; (f) i. 2,4,6-triisopropylbenzenesulfonyl chloride, pyridine, 40 °C; ii. 6; (g) NaIO4, MeOH/H2O, 0 °C.

consisted of HPLC grade solvents A: 0.1% formic acid in H2O and B: 0.1% formic acid in MeCN. Modified peptides were eluted initially 0% B for 10 min, then to 2% B for 5 min, then to 25% B for 35 min, to 90% B for 15 min, remaining for 9 min, then to 5% B for 1 min, then to 0% B for 5 min, and remaining for 10 min. Most of the adducts showed in MS2 the loss of the biotinylated glyceryl-phosphate (543 amu) and the precursor of the modified AcAVAGKAGAR. Further MS3 of the modified peptide precursor gave a series of b and y ions with modifications at lysine. Most of the adducts were found as doubly charged species in full MS. In MS2, they gave fragmentations indicative of the biotinylated glyceryl-phosphates and the corresponding modified peptide precursors to be carried on to MS3 to give appropriate b and y ions. Modification of HSA by PLPBSO. HSA (1 mg/mL) and POPC/ PLPBSO (5:1) were mixed and sonicated for 10 min to form the liposome. Then the reaction mixture was oxidized at 37 °C for 5 h, initiated by the water soluble azo initiator AIPH (final concentration, 1 mM). Adducts were reduced with NaCNBH3. The reaction mixture was concentrated using Amicon (MWCO: 30,000). The concentrated reaction mixture was applied to a monomeric avidin column. Plasma Oxidation and Identification of Biotinylated Human Serum Albumin in Plasma. PLPBSO supplemented plasma (50 µL) was oxidized using 10 mM AIPH overnight at 37 °C. Adducts were stabilized by reducing with 100 mM NaCNBH3 for 1 h at room temperature. The diluted plasma (1:10 ) plasma/PBS) was added to 50 µL bed volumes of streptavidin beads, which was incubated at room temperature for 2 h with rotation to allow biotinstreptavidin binding to take place. The beads were then washed 3 × 500 µL of 6 M urea in PBS, followed by 3 × 500 µL of 1 M NaCl in PBS and again with 3 × 500 µL of 6 M urea in PBS. These stringent washings conditions were used to limit the amount of nonspecific protein binding to the streptavidin beads. The biotinylated proteins were eluted using 100 µL of 1 M formic acid in 25% acetonitrile/75% H2O overnight at room temperature with rotation. Crude, flow through and eluted plasma proteins were resolved by SDS-PAGE on a 10% NuPAGE Novex Bis-Tris gel

and transferred onto a PVDF membrane. The membrane was then incubated with Streptavidin Alexa Fluor 680 conjugate or a rabbit polyclonal antibody to HSA and was imaged using the LI-COR Odyssey imaging system. UV and MALDI Analyses of Biotinylated HSA. Every 500 µL of flow through and elution was collected in disposable UV cuvettes and the absorbance measured at 280 nm. UV active flow through and elution fractions were combined and dried in speed vacuum. They were resuspended in 100 µL of H2O. Then, 10 µL was desalted using zip tips prior to MALDI analysis. Accelerating voltage was 25,000 V, and extraction delay was 150 ns, operated in positive and linear mode with a laser intensity of 1900.

Results and Discussion Synthesis. The design of the modified glycerylphosphatidylcholine is based on the notion that replacement of one of the methyl groups of the trimethylammonium headgroup with an ethylene glycol chain represents a minimal perturbation on the basic structure. The ethylene glycol arm would thus provide a means for attachment of a biotin recognition group that could be used in association with avidin/streptavidin capturing techniques. In a similar manner, Regen and co-workers have linked two glycerophosphatidylcholines at the choline headgroup with an alkyl disulfide group (17). These workers report that the dimeric phosphatidylcholines have packing behavior, melting temperatures, and monomer unit structures that mimic those of corresponding PCs. These compounds also undergo ideal mixing with physiologically relevant membrane fluid phases. We note that phosphatidylcholines are particularly appropriate for our study because they are major constituents of animal cell membranes. The general synthesis of the biotin-modified phosphatidylcholines is outlined in Scheme 1. All of the steps from 1 to key intermediate 6 occur in good to excellent yield. All intermediate

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Figure 1. Electron density profiles at ambient temperature for fully hydrated DPPB (top) and DPPC (bottom). The high-density peaks centered at +15-20 Å in each profile correspond to the lipid headgroup region.

compounds in the sequence have been purified and fully characterized. Coupling (17) of 6 with the appropriate glycerylphosphatidic acid gives PLPB and DPPB with yields that are highly variable. Our best experiments gave yields for the transformation that approached 60%, but more typically the yields were poor, between 10 and 20%. This reaction was carried out a number of times under a variety of conditions, but we could not identify the source of the problem in the transformation. In spite of this poor reaction, we could nevertheless prepare product biotinylated phospholipids on a 1 to 200 mg scale, making extensive studies of these compounds possible. The conversion of the PLPB to the oxidized version (PLPBSO) can be achieved in good yield by sodium periodate oxidation of the biotin tetrahydrothiophene unit to the corresponding sulfoxide. X-ray Diffraction. DPPB, the biotinylated phosphatidylcholine analogue of 1,2-dipalmitoyl-glycerylphosphatidyl-choline, DPPC, was studied by X-ray diffraction as both fully hydrated liposomes and partially hydrated multilayers. At ambient temperature (19), both preparations gave a lamellar low-angle pattern with 4 orders of a repeat period of 58 Å. The wideangle spacing was a single, sharp reflection with a spacing of 4.1 Å. In comparison, fully hydrated DPPC gave a lamellar repeat of 64 Å and a wide-angle pattern containing a sharp reflection at 4.2 Å and a broadband at 4.1 Å. Electron density profiles of DPPB and DPPC are presented in Figure 1. For each, the hydrocarbon regions of the bilayer are centered at the origin, the highest density peaks (at (16 Å for DPPB and at (21 Å for DPPC) correspond to the phosphate headgroups, and the medium-density regions at the outer edge of the profiles correspond to the fluid-containing regions between adjacent bilayers. The difference in location of the phosphate peaks indicates that bilayer thickness is about 10 Å greater for DPPC than for DPPB, and the higher electron densities at the outer edges of the DPPB profiles show the presence of the biotin functional group. For DPPC, the middle of the hydrocarbon core of the bilayer (at 0 Å) contains a sharp electron density trough due to the localization of the low-density terminal methyl groups (20, 21). In contrast, there is no trough in the middle of the bilayer for DPPB. These X-ray data indicate that at 20 °C both

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DPPB and DPPC form gel phase bilayers. The observed differences in lamellar spacing, wide-angle reflections, and electron density in the center of the profiles can be explained by differences in the hydrocarbon chain organization of the two systems. DPPC forms bilayers with tilted hydrocarbon chains (22, 23), whereas the profiles and wide-angle data for DPPB are the same as those of the bilayers where the gel hydrocarbon chains are oriented perpendicular to the bilayer surface but interpenetrate or interdigitate across the middle of the bilayer (24, 25). The X-ray diffraction analysis shows that DPPB forms interdigitated bilayer structures similar to those formed from DPPC in the presence of a number of surface active molecules, such as methanol, benzyl alcohol, ethylene glycol, and glycerol (24). All of these molecules displace water from the interfacial region, but they do not extend deeply into the bilayer interior, apparently anchoring to the interface by virtue of their polar moiety, with the nonpolar part of the molecule intercalating between the gel state hydrocarbon chains. The compounds cause the interpenetration of apposing lipid hydrocarbon chains for DPPC, reducing bilayer thickness. We suggest that the biotin ethylene-glycol headgroup of DPPB causes the same aggregate perturbations on the bilayer structure as do small molecule amphiphiles, resulting in an interdigitated bilayer structure for this lipid (Figure 1). It seems likely that other phosphatidylcholines modified with amphiphilic groups at the ammonium headgroup, such as Regen’s linked dimers, might also form similar bilayer structures. Peroxidation Reactions. Thin-film emulsions of PLPC (1palmitoyl-2-linoleoylglycero-phosphatidylcholine) and analogues PLPB and PLPBSO were oxidized by exposure to the water-soluble azo free radical initiator 2,2′-azobis[2-(2-imidazolin-2-yl)-propane] hydrodichloride (AIPH). Oxidation mixtures were shown by reverse-phase HPLC/MS analysis to be complex. A number of compounds in which the linoleate group had undergone apparent oxidative cleavage to aldehydes were detected (Figure 2). In the AIPH-initiated oxidation of PLPB, a product identical to the sulfoxide, PLPBSO, was formed in the free radical oxidation reaction, and very little linoleate chain fragmentation products such as the oxo-nonanoate (ONA) were observed from this precursor. However, the oxidation of PLPBSO, in which biotin is already oxidized to the sulfoxide, led to a set of products similar to those observed from PLPC. Among the products identified by HPLC/MS from the oxidation of PLPC and PLPBSO are the phospholipid esters KODA, keto oxo-dodecenoate; HODA, hydroxy oxo-dodecenoate; ONA, oxo-nonanoate; ODA, oxo-decanoate; OUA, oxo-undecenoate; and EOLA, epoxy oxo-linoleate. The observation that during the free radical oxidation of PLPB little linoleate chain fragmentation occurs to reactive electrophiles suggests to us that PLPB hydroperoxide oxidation products serve to oxidize the tetrahydrothiophene unit in the biotin headgroup (Scheme 2) (26). Biotin oxidation is concomitant with lipid hydroperoxide reduction, and it, therefore, serves to convert the reactive hydroperoxide intermediate to a stable endproduct, thus quenching the formation of electrophilic byproducts from hydroperoxide decomposition. Free radical oxidation of PLPBSO results in the normal electrophilic endproducts of linoleate chain fragmentation because the hydroperoxide products are not reduced by the cyclic sulfide and survive to undergo decomposition to electrophilic aldehydes. The biotin sulfoxide PLPBSO is, therefore, preferred for our studies because it oxidizes to give electrophilic products similar to those found from the natural phospholipid. Furthermore,

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Figure 2. Selected oxidation products from PLPC and PLPBSO.

Figure 3. Selected adducts of PLPC and PLPBSO with AcAVAGKAGAR. Primary fragmentations of either a or b is glyceryl-phosphate and its corresponding modified peptides precursors.

Scheme 2. Proposed Intermolecular Oxidation of Biotin Sulfur by Hydroperoxides

biotin-sulfoxide complexes to avidin and streptavidin with nearly the same avidity as that of biotin (27, 28), a requirement for the isolation of protein-phosholipid adducts. Peptide-Lipid Adducts. Liposome emulsions of PLPBSO and POPC (as the carrier) and the model peptide AcAVAGKAGAR were reacted in the air for 5 h at 37 °C with the free radical initiator AIPH. After reduction with sodium cyanoborohydride (NaCNBH3), the reaction mixture was chromatographed on a C18 reverse-phase column with a solvent gradient of 0.1% formic acid in water and acetonitrile. Products were monitored by a linear ion trap MS instrument equipped with a microelectrospray source. A number of compounds were detected, which appear to be covalent adducts of the peptide and phospholipid. Three of these adducts are shown in Figure 3, where R refers to either the PC or biotin side chain, and R′ is the lysine link to the peptide. The protonated species referred to as the HODA imine adduct b, has an MH+ m/z ) 1891, whereas MH+ of the corresponding Michael adduct b is observed at m/z ) 1909. In the case of MH+ of the HODA imine and Michael a, the adducts were found at m/z ) 1535 and 1551, respectively. Other species were observed with MH+ m/z values consistent with the structures of imine or Michael adducts of KODA, ONA, and ODA.

The parent phospholipid-peptide adducts undergo primary CID fragmentation at the glyceryl-phosphate bond, and subsequent MS3 on the peptide-containing fragment yields b and y ions consistent with an adducted lysine residue. Because of the high molecular weight of the modification, we encountered difficulties using established proteomics algorithms such as P-Mod (see discussion that follows) that are generally used to determine peptide adduction sites in these adducts. In subsequent experiments, the PLPBSO-AcAVAGKAGAR oxidation mixture was reduced with NaCNBH3 and mixed with streptavidin beads. After extensive washing of the beads with urea and HEPES buffer, the bound peptide-PLPBSO adduct was released from streptavidin by hydrolysis with 15% ammonium hydroxide over 15 h. The hydrolysate was then analyzed by LC-MS/MS on a linear trap MS instrument equipped with a microelectrospray source. A number of species corresponding to imine adducts to a linoleate-derived fatty acid were observed in P-Mod analysis. P-Mod is particularly useful in this application because the algorithm identifies MS/MS spectra of modified peptide forms even when the mass and sequence position of the modification is not anticipated. This allowed the detection of MS/MS spectra of several different adducts formed from PLPBSO oxidation products.

232 Chem. Res. Toxicol., Vol. 20, No. 2, 2007

Figure 4. MS2 spectrum (m/z ) 527 (M

2+

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+ H) of KODA imine. The y ions are shown in red, b ions in blue, and precursor ion in green.

Table 1. Partial P-Mod Output of the PLPBSO Derived Adducts on Model Peptide AcAVAGKAGAR after Base Hydrolysis peptide

P-val

mass shift

charge

precursor

peptide mass

position

scan number

AcAVAGKAGAR AcAVAGKAGAR AcAVAGKAGAR AcAVAGKAGAR

6.65E-06 9.61E-06 1.71E-03 1.78E-03

210.76 170.38 156.24 182.94

2 2 2 2

527.09 506.90 499.83 513.18

841.40 841.40 841.40 841.40

5 5 5 5

7562 5948 5450 6123

Table 2. Major Lysine Adducts Found in the Reaction with AcAVAGKAGAR and Oxidized PLPC, PLPBSO, Linoleic Acid, and PLPBSO Followed by Base Hydrolysis in 15% NH4OHa Lys modification

PLPC MH+, found

PLPBSO MH+, found

KODA

1549 (M) 1533 (I)

1907 (M) 1889 (I)

1071 (M) 1054 (I) 1036 (P)

HODA

1551 (M) 1535 (I)

1909 (M) 1891 (I)

1071 (HA) 1038 (P)

ONA

1476 (I)

1835 (I)

nd

999 (I)

ODA

1490 (I)

1852 (I)

1013 (I)

1012 (I)

OUA

1523 (M) 1504 (I)

nd nd

1043 (M) 981 (I-CO2)

1026 (I)

EOLA

1632 (M)

1991 (M)

linoleic acid MH+, found

nd

PLPBSO/NH4OH MH+, found 1069 (HA) 1053 (I) nd nd

nd

a

M, Michael adduct; I, imine; P, pyrrole; HA, hemiacetal; nd, not detected; KODA, keto oxo-dodecenoic acid; HODA, hydroxy oxo-dodecenoic acid; ONA, oxo-nonanoic acid; ODA, oxo-decanoic acid; OUA, oxo-undecenoic acid; EOLA, epoxy oxo-linoleic acid.

A typical P-Mod output is shown in Table 1. The first line of Table 1 shows the AcAVAGKAGAR peptide with a mass shift of 210.7 found on position 5 with a p-value of 6.65E-6, which indicates a KODA imine modification at the lysine residue with a highly significant p-value. The p-value reflects the probability of a false positive sequence-to-spectrum match. We also observed the ODA imine, ONA imine, and OUA imine adducts with mass shifts of 170.4, 156.2, and 182.9, respectively. Figure 4 shows a MS/MS spectrum of the KODA imine adduct on the AcAVAGKAGAR peptide. All b and y ions are consistent with a lysine-imine product of the 11-carbon-keto enal derived from linoleate with the AcAVAGKAGAR peptide. This same adduct was observed in the analysis of an oxidation reaction of linoleic acid with the model peptide. In Table 2, proposed adducts of AcAVAGKAGAR and electrophiles formed in the oxidation of PLPC, PLPBSO, and linoleic acid are presented. Also shown in the Table are species formed from

PLPBSO oxidation followed by base hydrolysis of the adducts with 15% ammonium hydroxide. Human Plasma and Serum Albumin. Liposome emulsions of PLPBSO and POPC (as carrier) and human serum albumin (HSA) were oxidized with AIPH as described above. Adducts were reduced with NaCNBH3 and applied to a monomeric avidin column. After extensive washing (flow-through) of the avidin column with PBS, biotinylated protein was eluted (release) with a 2 mM biotin/PBS solution. MALDI analysis of the flowthrough fractions showed a typical spectrum of HSA centered at m/z ) 66209. The elution fractions from the avidin column showed a broad MALDI peak between m/z ) 66,000 and 70,000, centered at m/z ) 67,600 (data not shown), a result that is consistent with significant adduction of lipid to the protein. The interest in these biotinylated phospholipids ultimately lies in their potential to identify phospholipid-adducted proteins

Phospholipid Peroxidation-Protein Adducts

Chem. Res. Toxicol., Vol. 20, No. 2, 2007 233

Acknowledgment. We thank Lisa M. Manier at Vanderbilt University Mass Spectrometry Research Center for assistance with MS measurements and Professor Larry Marnett for helpful discussions. This work was supported by National Institutes of Health Grants NIEHS P01 ES013125, GM15431, GM27278, P30 ES00267, and NSF CHE 0407697. Supporting Information Available: Detailed syntheses of all other compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

References

Figure 5. Plasma-PLPBSO supplemented and non-supplemented oxidations initiated with AIPH. (A) Crude PLPBSO-plasma oxidation mixture. Only biotinylated proteins are visualized with the Streptavidin Alexa Fluor 680 conjugate. The non-supplemented sample gives a blank. (B) Crude, flow through, and eluted solutions from the Streptavidin pull down experiment. Only HSA was visualized with a rabbit polyclonal antibody against HSA and Alexa Fluor 680 goat antirabbit secondary antibody.

in biological fluids, tissues, or cells. We illustrate this strategy here with a preliminary report of plasma proteins that adduct to oxidized PLPBSO. Thus, we have carried out oxidation experiments following the addition of PLPBSO to fresh human plasma. This was done by adding a small volume of a concentrated PLPBSO solution in DMSO to plasma freshly separated from whole blood (final plasma concentration of PLPBSO of 200 µM and