the formation of oxidants is a hallmark of chemical toxicity , inflammation , and other types of environmental stresses . oxidative stress and oxidants also are involved in human diseases that account for significant morbidity and mortality , including cancer , atherosclerosis , and neurodegenerative diseases . although oxidative stress derives fundamentally from the excessive flux of reduced oxygen species , such as superoxide and hydrogen peroxide , secondary products of lipid oxidation may play critical roles in oxidant - associated molecular pathologies . lipid electrophiles such as malondialdehyde , hydroxyalkenals , oxoalkenals , epoxyalkenals , and -ketoaldehydes readily react with proteins , and the formation of covalent adducts is hypothesized to contribute to oxidant - related diseases and toxicities . because they reflect the occurrence of oxidative stress , protein adducts from lipid electrophiles most studies of protein modification by lipid electrophiles have examined a few prototypical lipid oxidation products , such as 4-hydroxynonenal ( hne ) , 4-oxononenal , and -ketoaldehydes ( levuglandins ) using mass spectrometry ( ms ) . this published work has identified relevant protein adduction chemistries with amino acid side chain nucleophiles and in some cases mapped adducts to specific sequences in modified proteins . collectively , the adduct mapping data indicate significant site - specificity in modification , which is further reflected by a recent analysis of the kinetics of competing alkylation reactions of hne with human serum albumin . polyunsaturated fatty acids in living systems are largely present as phospholipid esters and recent studies have focused on novel biological properties of phospholipids bearing epoxyisoprostane and epoxycyclopentenone substituents or a reactive ,-unsaturated carbonyl oxidation product at the sn-2 position . these products stimulate inflammatory signal transduction pathways in vascular endothelial cells , serve as ligands for the scavenger receptor cd36 , and facilitate lipid uptake in macrophages and the formation of foam cells . most of the implicated phospholipids have electrophilic substituents and could be expected to form covalent protein adducts . however , the identities of proteins covalently modified by phospholipid electrophiles have not been reported . a major challenge in studying protein damage by lipid electrophiles is the sheer diversity of products generated . for example , the oxidative decomposition of a phospholipid containing linoleate at the sn-2 position would yield several electrophilic phospholipid products . hne and 4-oxononenal are formed from the -end of linoleate ester , but other reactive electrophiles contain the carboxy - end of the linoleate ester . thus , ketooxododecenoate ( koda ) , hydroxyoxododecenoate ( hoda ) , and other adducts esterified to phospholipid are expected to be among the electrophile adducts formed with proteins from oxidized linoleate - containing phospholipids ( figure 1 ) . characterization of the protein targets of the family of electrophiles formed from a phospholipid is also complicated by the relatively low levels of adducts compared to unmodified proteins in relevant biological systems . analysis of the protein targets of a family of phospholipid electrophiles requires a means of high affinity capture of the adducted proteins . recently , we reported the synthesis of the biotinylated phosphatidylcholine plpbso , which is the biotin - sulfoxide analogue of 1-palmitoyl-2-linoleoylglycerylphosphatidyl - choline ( plpc ) . oxidation of plpbso formed koda , hoda , and other expected linoleate oxidation products and also formed the corresponding electrophile adducts with a model peptide . in that work , we also demonstrated feasibility of streptavidin capture of plpbso - adducted human serum albumin from incubation of the protein with oxidized plpbso . here , we describe the use of plpbso as a probe to identify major protein targets of oxidized phospholipids in human plasma . this approach identified apolipoprotein a1 ( apoa1 ) as the principal target of plpbso oxidation products and led us to map adducts from lipid oxidation products on this protein . we report the identification of adducts from several phospholipid electrophiles , including carboxy - terminal adducts . the concordance of apoa1 site - specific modification between exogenous hne and endogenous phospholipid electrophiles suggests that the identified targets may be preferred modification sites on apoa1 in oxidative stress in vivo . plasma samples were collected from healthy human donors as part of an irb - approved study . 2,2-azobis[2-(2-imidazolin-2-yl)propane]-dihydrochloride ( aiph ) was from wako chemicals ( osaka , japan ) , streptavidin sepharose beads were from ge healthcare ( piscataway , nj ) , and ammonium hydroxide was from sigma ( st . louis , mo ) . apoa1 antibody ( rabbit polyclonal antibody to the rlaeyhakateh peptide of apoa1 ) was from cayman chemical ( ann arbor , mi ) . rabbit polyclonal antibody against hne histidine michael adducts was from calbiochem ( san diego , ca ) . alexa fluor 680-conjugates streptavidin and all secondary antibodies were purchased from molecular probes ( carlsbad , ca ) . mass spectrometry grade trypsin ( trypsin gold ) was from promega ( madison , wi ) . whole blood from fasting , healthy subjects was collected in a 440 ml acd blood collection bag ( baxter ) containing 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 . plpbso was dissolved in dmso ( 50 mm ) and was added to plasma at a final concentration 100 m ( < 0.3% v ) . they were placed in 37 c oil bath and stirred for 1.5 h. half of the supplemented and native plasma were aliquoted into eppendorf tubes and stored at 80 c until further use . hdl was isolated by ultracentrifugation ( 1.063 g / ml < d < 1.12 g / ml ) from both native and supplemented plasma . native and plpbso supplemented plasma ( 50 l ) were oxidized with 5 mm aiph overnight at 37 c . a total of 50 l of pbs was added to the sample , and 50 l of protein - g agarose slurry then was used to remove igg side chain that nearly comigrates with apoa1 on sds - page . the supernatant was collected and filtered through amicon 10 kda molecular weight cutoff filter to remove small molecules residual azo initiator and reducing agent . the samples then were diluted to 300 l with 100 mm ammonium bicarbonate , treated with 10 mm dtt at 50 c for 30 min , and then with 20 mm iam in the dark at room temperature . streptavidin sepharose beads ( 100 l slurry ) were washed with 3  500 l of 6 m urea in pbs . the oxidized , reduced , and alkylated native or plpbso supplemented plasma samples were added to the washed streptavidin beads and allowed to rotate for 2 h at room temperature . the supernatant was discarded and the beads were washed with 3  500 l of 6 m urea in pbs with rotation for 15 min at room temperature , then with 3  500 l of 1 m nacl with rotation for 15 min at room temperature , and finally with 3  500 l of 50 mm ammonium bicarbonate . elution of biotinylated proteins was done by selective hydrolysis of the plpbso acyl ester using 100 l of 15% nh4oh in 3 m urea overnight at room temperature . after hydrolysis , the supernatant was collected and the beads were rinsed with 2  100 l of 50 mm ammonium bicarbonate and combined with the eluate , which was placed under house vacuum for 30 min to remove excess nh3 . samples were enzymatically digested for 1824 h at 37 c using 0.5 g of trypsin and the solution then was acidified to ph 3 with formic acid to halt the digestion . then , the samples were evaporated under vacuum , desalted using zip - tips ( millipore c18 , millipore , billerica , ma ) , and resuspended in 200 l of 0.1% formic acid for lc - msms analysis . lc - msms analyses were performed on a thermo ltq linear ion trap instrument ( thermo electron , san jose , ca ) equipped with a thermo surveyor hplc system , nanospray source , and microautosampler . peptides were resolved on a 100 m  11 cm fused silica capillary column ( polymicro technologies , llc , phoenix , az ) packed with 5 m , 300  jupiter c18 ( phenomenex , torrance , ca ) . liquid chromatography was carried out at ambient temperature at a flow rate of 0.2 ml min 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 approximately 2 kv . a gas - phase fractionation method 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 400600 , then m / z 600900 , and finally m / z 9002000 . five data - dependent msms scans were performed on the selected precursor ions from each full scan . msms spectra were recorded using dynamic exclusion of previously analyzed precursors for 30 s with a repeat of 1 and a repeat duration of 2 . msms data were evaluated using the sequest algorithm 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 parameters such as xcorr , rsp , and sp scores . in this case , both the native oxidized plasma ( control ) and plpbso supplemented oxidized plasma msms data were filtered using xcorr score thresholds of 2 for 1 + peptides , xcorr 2.5 for 2 + peptides , and xcorr 3 for 3 + peptides with rsp  5 and sp  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 adducted by plpbso . 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 supernatant was removed and the beads were rinsed with 2  100 l of 50 mm ammonium bicarbonate . 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 streptavidin and imaged using the licor odyssey imaging system ( licor , lincoln , ne ) . both oxidized and unoxidized plasma were rotated with 100 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 . plpbso supplemented oxidized plasma was incubated with 100 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 1215 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 ( 2528 kda ) was excised and subjected to in - gel tryptic digestion . the eluted tryptic peptide solution was evaporated to dryness and the peptides were resuspended in 50 l of 0.1% formic acid in water for lc - msms analysis . msms spectra were acquired as described above , except that gas - phase fractionation was not used ; precursor ions for msms were selected from full scans over the range m / z 3002000 . the data were analyzed with the p - mod algorithm using apoa1 tryptic peptide sequences to detect msms spectra corresponding to mass - modified apoa1 peptides . 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 ) . to characterize the formation of hne adducts in hne - treated samples , isolated hdl was treated with hne at concentrations of 1 m , 10 m , 100 m , and 1 mm . 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 2028 kda . to determine hne adducts formed endogenously during hdl oxidation , hdl was incubated with aiph initiator overnight at concentrations of 0 , 100 , 1000 , and 5000 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 - msms and the data then were searched with the p - mod . 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 reactive aldehydes , ,-unsaturated aldehydes , epoxy-,-unsaturated aldehydes , and other products that can form protein adducts that are stabilized with nabh3cn . oxidation products in which the electrophile comprises a remnant portion of the linoleate chain at the sn-2 position will covalently link the adducted protein to the biotinylated choline headgroup , which enables capture with streptavidin . thus , capture of biotinylated proteins following oxidation enables capture and analysis of the protein targets of plpbso - derived phospholipid electrophiles . we used nabh3cn to stabilize adducts formed with plpbso oxidation products in order to detect both hoda and koda adducts . although nabh4 traps michael adducts more efficiently , it also reduces koda ketones to hydroxyls . on the other hand , the latter reaction was sufficiently slow in our studies to potentially allow redistribution of adducts between michael and imine forms . model studies ( not shown ) with an hne - adducted peptide indicated that reduction with nabh4 for 4 h at room temperature yielded only the michael adduct , whereas reduction with nabh3cn under the same conditions yielded the reduced michael adduct and nonreduced aldehyde and imine adducts . because our product analyses were essentially qualitative in nature although digestion and lc - msms analysis of the captured proteins will enable their identification , the adduct peptides would most likely go undetected because the phospholipid would suppress ionization or induce fragmentations in the phospholipid moiety that would compete with peptide fragmentation , thus , precluding identification by database search of msms spectra . we developed a mild ammonia hydrolysis procedure that cleaves the phospholipid fatty acyl ester bonds with minimal concomitant hydrolysis of the adducted protein ( figure 1 ) . selective ammonia hydrolysis enabled release of the biotinylated proteins from the streptavidin beads and left proteinlipid electrophile adducts containing a carboxy - terminal group ( figure 2 ) . stringent wash of the streptavidin beads containing captured proteins with 6 m urea and 1 m nacl reduced nonspecific binding to the streptavidin . following a tryptic digest of the released proteins , both adducted and 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 . oxidation of plasma supplemented with plpbso was initiated using the water soluble free radical initiator aiph . the water soluble free radical azo initiator , aiph , was used to initiate a true free radical chain reaction . the use of the azo initiator allows the rate of oxidation to be readily controlled by adjusting the concentration of aiph and the temperature of the reaction . full - scan lc - ms chromatograms of the tryptic digests indicate that the streptavidin beads captured a significant amount of biotinylated protein in the plpbso - supplemented , oxidized plasma , as indicated by the strong ion current in the lc - ms of the corresponding tryptic digests ( figure s1a in supporting information ) . in contrast , very little protein was captured on streptavidin from the nonsupplemented , oxidized plasma samples ( figure s1b in supporting information ) . to identify proteins adducted by plpbso , these tryptic digests then were subjected to lc - msms analysis using a gas - phase fractionation procedure and the data then were searched against the ipi human protein database . the experiment was repeated four times and 21 proteins were confidently identified ( 2 + peptide identifications at 95% confidence ) . the proteins represented by the highest sequence coverage , reflecting those present in the plpbso - labeled protein pool in greatest abundance , are listed in table 1 ( a complete listing of identified proteins is provided in table s2 in supporting information ) . sequence coverage is defined as the fraction of the protein sequence accounted for by msms - identified peptides . to confirm apoa1 adduction using immunoblotting techniques , immunoblotting was done with either a polyclonal antibody raised against apoa1 peptides or alexa fluor 680-conjugated streptavidin . the anti - apoa1 immunoblot indicated that , after oxidation in both the plpbso supplemented plasma and native plasma , the apoa1 band is shifted to higher apparent molecular weight ( figure 3 ) . the alexa fluor 680-conjugated streptavidin immunoblot shows biotin labeling of the higher molecular weight band , which is due to the covalent adduction of phospholipid to the protein . oxidation induces a migration shift of apoa1 in sds - page analysis and streptavidin labels the plpbso - modified apoa1 protein . oxidized plasma was immunoblotted with polyclonal apoa1 antibody followed by alexa fluor 680 antirabbit ( left ) or with antistreptavidin ( right ) . to identify the adducts with carboxy - terminal electrophiles in apoa1 , plpbso - supplemented plasma was oxidized with aiph followed by nacnbh3 reduction to stabilize the adducts and plpbso - adducted proteins were captured with streptavidin beads . after stringent washes to remove nonspecifically bound protein , the beads were treated with 15% ammonium hydroxide to selectively hydrolyze the phospholipid esters . the eluted proteins ( enriched in carboxyl - terminal lipid adducts ) were resolved by sds - page and the apoa1 band was excised for in - gel trypsin digestion followed by lc - msms analysis . analysis of the msms data with the p - mod algorithm searched apoa1 tryptic peptide sequences against the msms data to identify peptides that correspond to apoa1 peptides , including adducts . this approach does not require the user to specify anticipated adduct masses or sequence specificities ( e.g. , his modification ) , but instead performs an unbiased search for spectra that match search sequences with or without mass shifts due to adducts . the program then maps adduct positions based on msms fragmentation and calculates probabilities of random matches . the apoa1 search sequences used included miscleaved peptides that may result from lysine adduction by electrophiles . our criteria for reporting adducts included ( 1 ) presence of b - and / or y - ion series consistent with the adducted peptide sequence , ( 2 ) localization of adduct mass shifts by p - mod to h or k residues capable of reacting with the electrophiles to form the indicated adducts , ( 3 ) presence of diagnostic water loss fragments formed from hydroxy - containing adducts ( e.g. , reduced michael adducts ) . this analysis mapped sites of apoa1 modification and the detected mass shifts enabled identification of the carboxylated electrophiles ( table 2 ) . annotated msms spectra of these adducts are provided in figure 5 and figure s2 in supporting information . the identified residues include both lysine ( k45 , k106 , k188 , k195 ) and histidine residues ( h135 , h155 , h162 ) and included both imine ( schiff base ) and michael adducts with carboxylated simple aldehydes , as well as carboxylated ,-unsaturated aldehydes ( hoda and koda ) . as noted above , the use of nabh3cn as a reducing agent enabled us to distinguish koda and hoda adducts . however , we note that koda adducts corresponding in mass to michael addition products may also be ketoamide adducts , which were recently described by sayre and colleagues and which would not be distinguished by our ms analyses . lc - msms of the t*hlapysdelr tryptic peptide sequence from apoa1 modified with koda ( a ) , with hne ( b ) at his 162 ; of the qkl*helqek tryptic peptide sequence modified with 7-oxoheptanoic acid at his135 ( c ) ; and of the leal*kenggar tryptic peptide sequence modified with 8-oxooctanoic acid at lys182 ( d ) . we note the formation of the apparently novel imine adducts of 7-oxoheptanoic acid , 8-oxooctanoic acid , and 9-oxononanoic acid in these analyses . the latter is formed by the hock cleavage reaction that also generates hne during linoleate oxidation . formation of 8-oxooctanoic acid likely proceeds by  fragmentation of the linoleate 9-hydroperoxide to give the 8-yl - octanoic acid . this radical would react with oxygen to give the 8-hydroperoxyoctanoic acid which , upon dehydration , would give the 8-oxooctanoic acid compound . these analyses also identified some electrophile adducts derived from -terminal portion of the phospholipid - bound fatty acyl chain . these detected mass shifts corresponded to schiff base adducts of hexanal , heptanal , octanal , and nonanal ( table s1 in supporting information ) . detection of these species was less consistent from experiment - to - experiment than was detection of the carboxylated adducts described above . this may reflect the fact that the streptavidin capture and wash protocol would select against -terminal adducts , unless apoa1 molecules modified by these species also were covalently labeled with the phospholipid . although adducts were found throughout the protein , the adduction of h162 is of interest because this residue is within a sequence known to interact with lecithin - cholesterol acyltransferase ( lcat ) , an enzyme that contributes to hdl particle maturation . our analyses of the streptavidin - captured apoa1 ( see above ) identified relatively few adducts corresponding to modification by noncarboxylated electrophiles originating from the -terminus of the fatty acid ( e.g. , hne ) . to determine if hne adducts are formed on apoa1 upon oxidation of native hdl associated lipids , hdl particles were isolated from plasma and then oxidized overnight with different concentrations of aiph or treated with hne . anti - hne immunoblotting revealed immunoreactivity of the apoa1 band ( 2026 kda ) from hdl particles oxidized with 5 mm aiph or treated with either 100 m or 1 mm hne ( figure 4a ) . the visible gel in figure 4b shows that a molecular weight shift and band broadening for apoa1 ( similar to that observed in figure 2 , above ) was induced by as little as 100 m aiph , but not by hne treatment . this suggests that the apoa1 migration is affected perhaps by multiple modifications of the apoa1 protein that affect protein charge , shape and/or sds binding , but that hne adduction alone does not produce these effects . 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 2026 kda is induced by oxidation , but not by hne . hne adduction sites on apoa1 were mapped by lc - msms analysis of in - gel tryptic digests of the apoa1 band from aiph - oxidized and hne - treated samples described above . msms spectra were acquired by data - dependent scanning mode , which automatically samples intact peptide ion signals for msms . the msms spectra were searched against a human protein sequence database with the sequest algorithm with allowance for variable modifications on histidine and lysine residues for borohydride - reduced hne schiff base adducts ( + 140 amu ) or michael adducts ( + 158 amu ) . as a complementary approach , the sequences of apoa1 tryptic peptides were searched against the msms data with the p - mod algorithm to enable detection of spectra corresponding to modified peptides . p - mod hits corresponding to the expected + 140 and + 158 mass shifts on histidine or lysine residues were recorded . all putative spectra of adducts were subjected to manual inspection to verify the quality of sequence matches . ten sites of adduction were identified , including k12 , k23 , k94 , k96 , k107 , h135 , h155 , h162 , h193 , h199 , k208 , and k238 ( table 3 ) . accurate mass measurements for the adduct precursor ions with a thermo ltq - orbitrap instrument confirmed calculated values to within 36 ppm ( table 3 ) . mass error ( ppm ) for measurements of the precursor ions of the observed peptide adducts were analyzed on a thermo ltq - orbitrap instrument and were as follows : k12 , 3.2 ppm ; k23 , 3.8 ppm ; k94 , 3.9 ppm ; k96 , 3.8 ppm ; k107 , 3.7 ; h135 , 4.0 ppm ; h155 , 3.2 ppm ; h162 , 2.9 ppm ; h193 , 3.7 ; h199 , 3.3 ; k208 , 3.8 ppm ; k238 , 5.9 . denotes that the adducts were detected in either 1 of 3 , 2 of 3 , or 3 of 3 independent experiments , respectively . in replicate experiments in which hdl was treated with either hne or oxidized with aiph , histidines in the sequences thlapysdelr and laeyhak were repeatedly found to be modified even with exposure to as little as 1 m hne ( table 3 ) . his162 , which lies within the lcat - interacting domain of apoa1 , was a target for modification by both hne and the carboxyated electrophile koda , representative msms spectra for which are both shown in figure 5 . although lipid electrophiles generated during oxidative stress are widely thought to play critical roles in injury and adaptation to stress , relatively little is known about the protein targets of these species . target identification is complicated by the great diversity of potential targets , the low abundance of modifications , and the difficulty of selectively capturing and analyzing the modified proteins . we have approached this problem by employing affinity - tagged lipid substrates that form oxidation products representative of the same lipid class , but that also enable high affinity capture of the lipid electrophile adducts . the plpbso probe generates oxidation products similar to the corresponding phosphatidylcholine analogue and also forms adducts to proteins . use of plpbso enabled detection of apoa1 as a major protein target of phospholipid electrophiles in plasma . subsequent analysis of apoa1 from oxidized plasma identified novel lipid electrophile adducts derived from phospholipid oxidation products . moreover , adduct mapping studies revealed adduction in a functionally critical region of the protein . apoa1 is the core protein component of hdl particle , which mediates reverse cholesterol transport from tissues to the liver and also exerts antiatherogenic effects . recent work indicates that hdl function is impaired in cardiovascular disease and systemic inflammatory disorders . functional impairment of hdl has been attributed to oxidative modifications of apoa1 by myeloperoxidase ( 4954 ) or reactive products of lipid oxidation . thus , our results indicating that apoa1 is a major target of reactive phospholipid electrophiles led us to focus attention on the types and site - specificity of apoa1 adduction . the biotin tag on the choline headgroup in plpbso allows affinity capture of protein adducts that contain the phospholipid headgroup . these adducts are derived from electrophiles esterified to the phospholipid and form carboxylated adducts upon ammonia hydrolysis . nevertheless , not all adducts identified from streptativin - captured protein are necessarily derived from plpbso . some of the identified adducts may originate from endogenous apoa1 lipid esters and are formed on apoa1 that is also adducted with plpbso . the formation of carboxylated lipid electrophile adducts has been studied to only a limited extent . williams and colleagues characterized koda and hoda adducts formed by decomposition of linoleate hydroperoxide in the presence of purified cytochrome c. we also observed adducts with 7-oxoheptanoic acid and 8-oxooctanoic acid on apoa1 lysine residues . although the formation of these imine adducts is not unexpected , this is the first report of the direct detection of these adducts , to our knowledge . the covalent binding of aldehyde - containing phospholipids has been shown previously to generate antigenic components of oxidized low density lipoprotein ( ldl ) . these changes , in turn , promote recognition of oxidized ldl by c - reactive protein and functional alterations in monocytes exposed to oxidized ldl . a notable characteristic of apoa1 subjected to free radical oxidation is the alteration of its migration characteristics in sds - page ( figure 3 ) . this may reflect the formation of carboxylated adducts on histidine and lysine residues , which would generate a variety of apoa1 forms with modified charge characteristics and possibly altered sds binding . in contrast , treatment with hne does not produce this effect , even when treatment produced comparable levels of hne adduction . our analyses of apoa1 adducts from plpbso - supplemented , oxidized plasma that was captured with streptavidin identified several carboxylated adducts , but relatively few adducts corresponding to electrophiles derived from the -terminus of the fatty acid ( e.g. , hne ) . on the other hand , apoa1 was modified by hne formed during oxidation of isolated hdl with aiph and by exogenously added hne ( table 3 ) . in both cases , the distribution of hne adducts was similar . this confirms that apoa1 can undergo modification both by diffusible hne - like electrophiles and phospholipid - bound electrophiles . the somewhat sporadic detection of hne and other noncarboxylated adducts in plpbso - supplemented plasma following streptavidin capture probably reflects loss of the nonphospoholipid adducted apoa1 proteins during the wash steps to remove nonbiotinylated proteins . this also implies that , under the conditions of our experiments , most apoa1 molecules bear a total of one or two adducts and that these are distributed between all the observed sites . had the protein been more heavily modified , one would have expected to consistently observe hne adducts and other noncarboxylated adducts as well as carboxylated adducts in the streptavidin - captured protein . we identified adducts in two target sequences that have been identified previously in studies of oxidized apoa1 . these sites are immediately adjacent to tyr192 , which was identified by the hazen and heinecke groups as sites of nitration and chlorination in apoa1 . the latter reaction is catalyzed by myeloperoxidase , which binds to this sequence in apoa1 in hdl . we also mapped koda and hne adducts to his162 , which resides within a sequence recently shown to be critical for interaction with lcat . tyr166 undergoes chlorination and nitration in atheromas in vivo and this modification blocks the activation of lcat by apoa1 , which is an obligatory step in hdl particle maturation . his162 is also a solvent - exposed residue in the lcat - activating loop that contains tyr166 ( 44 ) and this raises the possibility that his162 adduction may have functional consequences similar to those reported for tyr166 modifications . our studies demonstrate that his 162 and his193 are among the most easily detected adducts at low hne exposure concentrations in hdl ( table 3 ) . it might be tempting to conclude that this indicates high kinetic reactivity of these residues with electrophiles . however , detection in lc - msms analyses depends not only on relative levels of the adducts , but also on ionization efficiency of the adducted peptides . in a recent study of the kinetics of human albumin adduction at different sites by hne , we noted that the rate constant for formation of the most readily detected adduct ( his67 ) was over 10-fold lower than that for the most reactive site ( his242 ) , which was less readily detected in data - dependent lc - msms analyses . thus , we are planning additional studies of the kinetics of apoa1 modification in plasma and hdl to characterize the hierarchy of reactivity with lipid electrophiles . our results suggest that covalent adducts with lipid electrophiles can mechanistically link lipid peroxidation , oxidative stress and disease mechanisms in a similar manner . in addition , lipid electrophile adducts of apoa1 merit evaluation as potential biomarkers of oxidative stress in vivo .
reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress - related pathologies by covalently modifying proteins and affecting critical protein functions . 
 the difficulty of capturing and analyzing the relatively small fraction of modified proteins complicates identification of the protein targets of lipid electrophiles . 
 we recently synthesized a biotin - modified linoleoylglycerylphosphatidycholine probe called plpbso ( tallmanet al.chem . 
 res . 
 toxicol.2007 , 7 , 22723417305406 ) , which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins . 
 supplementation of human plasma with plpbso followed by free radical oxidation resulted in covalent adduction of plpbso to plasma proteins , which were isolated with streptavidin and identified by liquid chromatography - tandem mass spectrometry ( lc - msms ) . among the most highly modified proteins was apolipoprotein a1 ( apoa1 ) , which is the core component of high density lipoprotein ( hdl ) . 
 apoa1 phospholipid adduct sites were mapped by lc - msms of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts . 
 several carboxylated adducts formed from phospholipid - esterified 9,12-dioxo-10(e)-dodecenoic acid ( koda ) , 9-hydroxy , 12-oxo-10(e)-dodecenoic acid ( hoda ) , 7-oxoheptanoic acid , 8-oxooctanoic acid , and 9-oxononanoic acid were identified . 
 free radical oxidations of isolated hdl also generated adducts with 4-hydroxynonenal ( hne ) and other noncarboxylated electrophiles , but these were only sporadically identified in the plpbso - adducted apoa1 , suggesting a low stoichiometry of modification in the phospholipid - adducted protein . 
 both phospholipid electrophiles and hne adducted his162 , which resides in an apoa1 domain involved in the activation of lecithin - cholesterol acyltransferase and maturation of the hdl particle . 
 apoa1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress .