the 
 major reactive aldehyde produced during peroxidation of polyunsaturated 
 fatty acids , malondialdehyde ( mda ) , reacts with nucleophilic sites 
 of dna and protein to form oxopropenyl adducts . reaction of mda with 
 dna produces primarily 3-(2-deoxy--d - erythro - pentofuranosyl)pyrimido[1,2-]purine-10(3h)-one ( m1dg ) and lesser amounts of 2-oxopropenyl - deoxyadenosine 
 ( figure 1 ) . m1dg is mutagenic in vitro and in vivo , and 2-oxopropenyl - deoxyadenosine alters dna polymerase 
 activity . both species 
 can 
 react with da or dg , resulting in oxypropenylation of the base . the reaction of mda with protein 
 occurs predominantly at lysine 
 residues under physiological conditions . protein adduct formation is rudimentary , as the exact 
 targets and sites of adduction are largely unknown . however , a particularly 
 interesting effect of mda exposure in human cells is the inhibition 
 of nucleotide excision repair ( ner ) , which is associated with increased 
 sensitivity to the mutagenicity of ultraviolet light and benzo[a]pyrene dihydrodiolepoxide . ner is the primary process for repair of bulky dna adducts , and 
 in humans , ner deficiencies result in xeroderma pigmentosum , a spectrum 
 of disorders characterized by hypersensitivity to sunlight , neurological 
 degeneration , and a greatly increased incidence of cancer . although not verified experimentally , inhibition 
 of ner by mda was proposed to arise from loss of function of mda - modified 
 ner proteins . a potential mda target 
 is the ner scaffold protein , xeroderma pigmentosum complementation 
 group a ( xpa ) , which binds to the damaged dna substrate via a c - terminal 
 basic cleft comprising multiple lysine residues . xpa coordinates recruitment and assembly of the enzymes that repair 
 the damaged dna , and mutations in its dna - binding domain are associated 
 with the most severe clinical xp phenotypes . oxopropenyl adducts to dna and protein can also be formed 
 through 
 reaction with base propenal , a reactive aldehyde produced by oxidant - mediated 
 hydrogen abstraction from the 4-position of dna bases ( figure 1 ) . base propenals are approximately 100-fold more reactive to dna than 
 mda , and experiments in which escherichia coli strains of varying membrane composition were exposed to oxidants 
 demonstrated that base propenals are more important than mda as a 
 source of m1dg . currently , 
 very little is known about potential protein targets of base propenal 
 adduction ; however , as a highly reactive form of dna damage , base 
 propenals are ideally situated to oxopropenylate dna - binding proteins 
 such as xpa . this has led us to hypothesize that base propenal - mediated 
 protein damage contributes to toxicity and mutagenicity associated 
 with oxidative or electrophile stress . to test this hypothesis , a 
 comprehensive evaluation of base propenal s ability to directly 
 damage proteins adenine propenal is known to react with 
 glutathione via michael addition to form glutathionylpropenal and 
 a glutathione  adenine propenal cross - link , but , to date , no detailed investigation of the reaction 
 of any base propenal with amino acids or proteins has been reported . one approach to evaluate the protein adduction potential of an 
 electrophile is to identify all sites of modification and the structures 
 of those modifications using a model protein . a frequently used target 
 for this purpose is human serum albumin ( hsa ) . albumin is highly abundant and has affinity for a broad range of 
 ligands , a relatively long half - life , and the potential for noninvasive 
 sampling . thus , it is an ideal candidate for the global qualitative 
 and quantitative evaluation of exposure to electrophiles of endogenous 
 or exogenous origin ( the exposome ) . in fact , in a large multilaboratory 
 comparison of biomarkers of in vivo oxidant generation , 
 the level of carbonylated plasma protein ( i.e. , albumin ) was one of 
 only two markers that correlated with oxidant exposure and tissue 
 pathology , and it was the only marker that was effective longer than 
 6 h after exposure . to exploit albumin s 
 potential as a biomarker , however , requires a more complete understanding 
 of its major sites of adduction and determinants of their reactivity 
 to a range of electrophiles . for example , whereas much attention has 
 been directed to hsa s highly reactive cys-34 as a sensor of 
 oxidant damage , the major site of oxopropenylation 
 by mda is lys-525 . here , we report 
 the application of a recently described mass spectrometric 
 approach to identify adenine propenal 
 modifications of hsa and xpa . our data demonstrate that , like mda , 
 adenine propenal primarily reacts at protein lysine residues ; however , 
 the major site of attack on hsa is distinct from that of mda . we also 
 report that adenine propenal modifies lysine residues of xpa , including 
 residues within its dna - binding domain and residues reported to be 
 sites of regulation via sirt1-mediated deacetylation . biophysical analysis confirms 
 that modification of xpa by adenine propenal decreases its affinity 
 for damaged dna . n--acetyllysine , trichloroacetic acid ( tca ) , dithiothreitol , 
 iodoacetate ( sigma ultra ) , and nacnbh3 were obtained from 
 sigma - aldrich ( st . nmr 
 experiments were 
 acquired using a 14.0 t bruker magnet equipped with a bruker av - iii 
 console operating at 600.13 mhz . all spectra were acquired in 3 mm 
 nmr tubes using a bruker 5 mm tci cryogenically cooled nmr probe . chemical shifts were referenced internally to d2o ( 4.70 
 ppm ) , which also served as the h lock solvent . for 1d h nmr , experiments were acquired with presaturation in order 
 to suppress the residual h2o signal , and typical experimental 
 conditions included 32k data points , 13 ppm sweep width , a recycle 
 delay of 1.5 s , and 32 scans . for 2d h h cosy , presaturation was also performed , and experimental conditions 
 included a 2048  1024 data matrix , 13 ppm sweep width , recycle 
 delay of 1.5 s , and 8 scans per increment . the data were processed 
 using squared sinebell window function , symmetrized , and displayed 
 in magnitude mode . c hsqc experiments were acquired using a 1024  128 data matrix , 
 a j(c  h ) value of 145 hz , which resulted in 
 a multiplicity selection delay of 34 ms , a recycle delay of 1.5 s , 
 and 80 scans per increment along with garp decoupling on c during the acquisition time ( 150 ms ) . the data were processed using 
 a p/2 shifted squared sine window function and displayed with ch / ch3 signals phased positive and ch2 signals phased 
 negative . c hmbc experiments were acquired using 
 a 2048  128 data matrix , a j(c  h ) value 
 of 9 hz for detection of long - range couplings resulting in an evolution 
 delay of 55 ms , j1(c  h ) filter 
 delay of 145 hz ( 34 ms ) for the suppression of one - bond couplings , 
 a recycle delay of 1.5 s , and 144 scans per increment . the hmbc data 
 were processed using a p/2 shifted squared sine window function and 
 displayed in magnitude mode . compounds were greater than 90% pure , 
 and mass spectral analysis was accomplished on an applied biosystems 
 3200 q trap mass spectrometer ( mds sciex ) . the mass spectrometer was 
 equipped with an electrospray source and operated in positive ion 
 mode . h nmr ( dmso - d6 ) :  ( ppm ) 7.17 ( dd , j1 = 7.88 hz , j2 = 14.4 hz , 
 1h ) , 7.55 ( br s , 2h ) , 8.27 ( s , 1h ) , 8.35 ( d , j = 
 14.4 hz , 1h ) , 8.63 ( s , 1h ) , 9.67 ( d , j = 7.88 hz , 
 1h ) . c nmr ( dmso - d6 ) :  
 ( ppm ) 119.2 , 120.5 , 136.4 , 142.8 , 147.6 , 151.4 , 158.2 , 190.8 . h nmr 
 ( d2o ) :  ( ppm ) 9.15 ( d , j = 10.0 
 hz , 2h ) , 5.73 ( t , j = 10.0 hz , 1h ) . the sodium salt of 
 malondialdehyde ( mda ) ( 100 mg , 1.06 mmol ) was added to a solution 
 of n--acetyllysine ( 200 mg , 1.06 mmol ) in 
 5 ml of ammonium acetate buffer ( ph 4.5 ) . the reaction was then stirred 
 at room temperature for 15 h. the reaction mixture was lyophilized , 
 and the residue was purified by column chromatography ( etoac / meoh , 
 5:1 ) on silica gel ( 60  porosity , 4063 m particle 
 size ) . h nmr ( d2o ) :  ( ppm ) 1.33 ( m , 
 2h ) , 1.561.61 ( m , 3h ) , 1.71 ( m , 1h ) , 1.93 ( s , 3h ) , 2.91 ( t , j = 7.3 hz , 2h ) , 4.05 ( m , 1h ) , 5.31 ( m , 1h ) , 7.44 ( d , j = 13.0 hz , 1h ) , 8.59 ( d , j = 8.5 hz , 
 1h ) . c nmr ( d2o ) :  ( ppm ) 22.0 , 23.4 , 
 27.2 , 32.0 , 39.3 , 55.3 , 86.6 , 163.7 , 173.7 , 179.1 , 191.2 . the sodium salt of mda ( 60 mg , 0.319 mmol ) was added to 
 a solution of n--acetyllysine ( 15 mg , 0.159 
 mmol ) in 2 ml of anhydrous methanol . the mixture was heated at reflux , 
 and the progress of the reaction was monitored by uv  vis spectroscopy . the product was then purified by column chromatography ( etoac / meoh , 
 5:1 ) on silica gel ( 60  porosity , 4063 m particle 
 size ) and hplc . hplc chromatography was carried out on a luna c18 
 column ( 250  4.60 mm , 5 m ) at a flow rate of 1.25 ml / min . the eluting solvents included solvent a ( 10 mm ammonium acetate ) and 
 solvent b ( acetonitrile ) . the following solvent gradient was used : 
 010.5 min linear gradient from 5% b to 20% b , followed by 
 a 2 min linear gradient to 80% b , hold for 6.50 min at 80% b , followed 
 by a 1 min linear gradient to the initial conditions of 5% b. the 
 product was observed with a retention time of 6.1 min . h nmr ( d2o ) :  ( ppm ) 1.30 ( m , 4h ) , 1.591.61 
 ( m , 6h ) , 1.73 ( m , 2h ) , 1.88 ( s , 6h ) , 3.22 ( m , 2h ) , 3.30 ( t , j = 7.3 hz , 2h ) , 4.08 ( m , 2h ) , 5.45 ( m , 1h ) , 7.49 ( d , j = 11.5 hz , 1h ) , 7.56 ( d , j = 11.5 hz , 
 1h ) . c nmr ( d2o ) :  ( ppm ) 21.3 , 21.8 , 
 25.7 , 30.2 , 42.0 , 53.7 , 88.5 , 158.9 , 160.5 , 177.5 , 178.9 . n--acetyllysine 
 ( various concentrations ) was mixed with 10 mm adenine propenal in 
 50 l reaction mixtures containing 10 mm nacl and 10 mm 4-morpholinepropanesulfonic 
 acid ( mops ) ( ph 6.5 ) . at 1 h intervals , aliquots of each reaction 
 mixture were injected onto a waters hplc system equipped with a 717 
 plus autosampler , 1525 binary hplc pump , and 2996 photodiode array 
 detector . analytes were separated using a phenomenex polar - rp synergi 
 hplc column , 75 cm  2 mm with 4 m particle size , at a 
 flow rate of 0.6 ml / min . the mobile phase solvents were h2o + 0.05% acetic acid ( v / v , solvent a ) and 3:1 meoh / acn + 0.05% acetic 
 acid ( v / v , solvent b ) delivered in a 19 min gradient consisting of 
 the following : 010.5 min , 520% b ; 10.512.5 
 min , 2080% b ; 12.516 min , 80% b ; 1619 min , 
 5% b. data were analyzed using empower software , and peak areas were 
 manually integrated . reactions 
 were performed 
 as described above and analyzed using a thermo accela hplc pump ( thermo 
 fisher scientific ) and htc pal autosampler ( leap technologies ) in - line 
 with a thermo quantum triple quadrupole mass spectrometer ( thermo 
 fisher scientific ) . the quantum was equipped with an electrospray 
 source operated in q1 full scan , positive ion mode . q1 was scanned 
 from 100600 amu in 0.45 s. analytes were chromatographically 
 separated on a synergi polar - rp hplc column ( phenomenex , 7.5  
 0.2 cm , 4 m particle size ) using the following gradient : 010.5 
 min , 520% b ; 10.512.5 min , 2080% b ; 12.516 
 min , 80% b ; 1617 min , 5% b. the mobile phase solvents were 
 h2o + 0.05% acetic acid ( v / v , solvent a ) and acetonitrile 
 + 0.05% acetic acid ( v / v , solvent b ) , and they were delivered at a 
 flow rate of 0.6 ml / min . all data were acquired and analyzed using 
 thermo xcalibur software . solutions containing 
 known concentrations of synthetic 3 and 4 were used to measure extinction coefficients of each compound . the 
 value obtained for both compounds ( 33  600  200 mcm ) was the same within experimental 
 error ; however , the wavelengths of maximal absorbance were different 
 ( 280 nm for 3 and 300 nm for 4 ) ( figure s1 ) . adenine propenal ( 10 mm ) was 
 incubated with increasing concentrations of n--acetyllysine 
 ( 1 , 2 , 5 , 10 , 50 , and 100 mm ) in reaction mixtures containing 10 mm 
 nacl and 10 mm mops ( ph 6.5 ) . the mixtures were incubated for 2 h 
 at room temperature and then diluted 1:1000 in h2o . the 
 absorbance at 300 nm of each dilution was then obtained using a du800 
 spectrophotometer ( beckman coulter ) , and the experimental extinction 
 coefficient of 4 was used to estimate its concentration 
 in the reaction mixture . note that , for reaction mixtures containing 
 > 10 mm n--acetyllysine , complete consumption 
 of adenine propenal eliminates concerns of its contribution to the 
 absorbance of the final reaction mixture . the adenine product does 
 not absorb significantly at 300 nm ( figure s1 ) . on the basis of its full uv spectrum ( figure 
 s1 ) , the extinction coefficient of 3 at 300 nm 
 is estimated to be 7.8  10 m cm . the presence of this product at a concentration 
 equal to approximately 10% of the concentration of 4 , 
 as found in the reaction mixtures , indicates that its contribution 
 to the absorbance at 300 nm will be no more than 3% . consequently , 
 the absorbance at 300 nm can be used to estimate the concentration 
 of 4 with reasonable accuracy . n--acetyllysine 
 ( 10 mm ) was incubated with adenine propenal ( 10 mm ) in 10 mm mops 
 ( ph 6.5 ) containing 10 mm nacl in a 200 l reaction mixture 
 for 30 min . after incubation , 50 mm nacnbh3 ( in h2o ) ( sigma - aldrich ) was added for an additional 30 min , and samples 
 were then analyzed using lc - ms as described below . lc - ms analysis 
 was performed on the thermo quantum triple quadrupole 
 mass spectrometer . here , the instrument was operated in q1 full - scan 
 negative ion mode using electrospray ionization . q1 was scanned over 
 the range 100400 amu over 0.5 s. chromatography was carried 
 out on a synergi max - rp column ( phenomenex , 7.5  0.2 cm , 5 m 
 particle size ) using the following gradient : 05 min linear 
 gradient from 2% b to 50% b , 57 min at 50% b , 710 
 min 50% b to 2% b. the mobile phase solvents were h2o + 
 0.05% formic acid ( v / v , solvent a ) and 3:1 ch3cn / meoh + 
 0.05% formic acid ( v / v , solvent b ) , and they were delivered at a flow 
 rate of 400 l / min . all data were acquired and analyzed using 
 thermo xcalibur software . using this chromatographic system , the reduced 
 intermediate eluted at approximately 2.9 min . n - acetylcysteine ( 200 
 m ) was combined with 50 m adenine propenal in 50 mm 
 sodium phosphate buffer , ph 7.4 , and incubated for 6 h at 37 c . nabh4 ( 50 mm ) 
 ( sigma - aldrich ) was used to reduce samples as indicated . analytes 
 were separated on a supelco ascentis c18 column ( 50  2.1 mm , 
 3 m ) with buffers a and b comprising 0.1% formic acid in water 
 and 0.1% formic acid in acetonitrile , respectively . the gradient was 
 as follows : 13 min , 1% b ; 37 min , 98% b ; 711 
 min , 98% b ; 1117 min , 1% b. samples were analyzed on a thermofinnigan 
 tsq quantum mass spectrometer with esi source interfaced to a thermofinnigan 
 ms pump plus and autosampler plus ( thermo , san jose , ca ) . n - acetylcysteine - adducted species were analyzed with data - dependent 
 scanning enabled in negative ion mode , scanning from m / z 150650 over 0.5 s. the top two m / z peaks from each ms scan were fragmented 
 with 10 ev collision energy for 0.5 s. hsa ( 15 m ) ( abcam , 
 cambridge , ma ) was incubated with increasing 
 amounts of adenine propenal ( 0.15 , 0.38 , 1.5 , 3 , and 7 mm ) , representing 
 a molar excess of adenine propenal to hsa of 10 , 25 , 100 , 250 , and 
 500 , respectively . reactions were incubated for 6 h at room temperature 
 in a buffer containing 10 mm nacl and 10 mm mops ( ph 6.5 ) . following 
 incubation , samples were precipitated with 25% trichloroacetic acid 
 ( sigma - aldrich ) on ice for 1 h , washed with cold acetone , dried , and 
 reconstituted in 50 mm tris ( ph 8.0 ) containing 50% 2,2,2-trifluoroethanol 
 ( acros organics , pittsburgh , pa ) . samples were reduced with dtt ( sigma - aldrich ) , 
 carbamidomethylated with iodoacetamide ( sigma - alrich ) , and diluted 
 5-fold with 100 mm tris ( to obtain a final solution containing 10% 
 2,2,2-trifluoroethanol ) . following dilution , samples were digested 
 with sequencing - grade trypsin ( promega , madison , wi ) overnight , acidified , 
 and diluted in 0.1% formic acid . the resulting solutions of trypsin - generated 
 peptides were loaded onto a capillary reverse - phase analytical column 
 ( 360 m o.d . 100 m i.d . ) using an eksigent nanolc 
 ultra hplc and autosampler . the 20 cm analytical column was directly 
 packed into a laser - pulled emitter tip using jupiter c18 reverse - phase 
 medium ( 3 m beads , 300  pore size , phenomenex ) . mobile 
 phase solvents consisting of 0.1% formic acid in water ( solvent a ) 
 and 0.1% formic acid in acetonitrile ( solvent b ) at a flow rate of 
 500 nl / min were used to elute the peptides . the 90 min gradient consisted 
 of the following : 010 min , 2% b ; 1050 min , 235% 
 b ; 5060 min , 3595% b ; 6065 min , 95% b ; 6570 
 min 952% b ; 7090 min , 2% b. mass analysis of eluting 
 peptides was performed on an ltq orbitrap xl mass spectrometer ( thermo 
 scientific ) , equipped with a nanoelectrospray ionization source with 
 detection in positive ion mode . the ltq orbitrap was operated using 
 a data - dependent method , enabling dynamic exclusion . full scan ( m / z 4002000 ) spectra were acquired 
 with the orbitrap ( resolution 60  000 ) , and the five most abundant 
 ions in each ms scan were selected for fragmentation in the ltq . an 
 isolation width of 2 m / z , 30 ms 
 activation time , and collision energy normalized to 35% were used 
 to generate ms spectra . dynamic exclusion settings allowed 
 for a repeat count of 2 within a 10 s duration , with exclusion duration 
 time set to 15 s. for identification of modified peptides , tandem 
 mass spectra were searched with sequest ( thermo fisher scientific ) 
 against a human subset database created from the uniprotkb protein 
 database ( www.uniprot.org ) . variable modifications of + 57.0214 
 on cys ( carbamidomethylation ) , + 15.9949 on met ( oxidation ) , and + 54.0105 
 on lys or cys ( oxypropenylation ) were included for database searching . search results were assembled with scaffold 3.0 ( proteome software ) 
 using threshold filtering criteria consisting of 95% peptide probability 
 to achieve a peptide false discovery rate estimate of 0.2% . sites of modification were validated by manual interrogation of tandem 
 mass spectra using xcalibur 2.1 qual browser software ( thermo scientific ) . calculations of mass errors ( in ppm ) of precursor ions mass analyzed 
 in the orbitrap were performed using the following equation : ( ( theoretical 
 mass  observed mass)/theoretical mass )  10 . to obtain theoretical masses , analysis of cross - links was performed 
 in a similar manner with inclusion of + 36 on lys for database searching . purified hsa ( 0.75 m ) was incubated with increasing concentrations 
 of adenine propenal in reaction mixtures containing 10 mm nacl and 
 10 mm mops ( ph 6.5 ) for 6 h at room temperature . following incubation , the protein was heated at 95 c for 10 min , cooled to room temperature , 
 and then treated with 1 mg of pronase ( calbiochem , gibbstown , nj ) 
 per mg of protein at 37 c for 24 h. pronase was inactivated 
 by heating at 95 c for 10 min , followed by cooling to room temperature , 
 and treating with 1 l of aminopeptidase m ( calbiochem , gibbstown , 
 nj ) at 37 c for 24 h. a final incubation at 95 c for 10 
 min inactivated the aminopeptidase m , and the samples were then cooled 
 to room temperature in preparation for adduct purification . for use as an internal standard , [ h]-[c]-4 was prepared by reaction 
 of mda with [ h]-lysine containing [ c]-lysine 
 as a tracer and quantified using a method similar to that described 
 for the [ h]-[c]-lysine - lactam internal standard 
 as described . an oasis hlb cartridge 
 ( 1 cm ) ( waters , milford , ma ) was equilibrated with 2 ml 
 of methanol followed by 2 ml of h2o . following addition 
 of [ h]-[c]-4 , the sample was 
 loaded onto the cartridge using gravity flow . the cartridge was washed 
 with 1 ml of h2o , and 4 was eluted with 3 
 ml 1:1 methanol / ethyl acetate and dried to 100 l under n2 gas . the sample was then diluted in 0.1% formic acid solution 
 to a final volume of 1 ml and filtered using a 0.22 m nylon 
 spin - x centrifuge tube spun at 6000 rpm for 5 min . samples were 
 purified using a thermo scientific aquasil c18 5 m reverse - phase 
 column using liquid scintillation counting to monitor elution of the 
 radioactive [ h]-[c]-lysine  lysine 
 cross - link standard . the internal standard [ h]-[c]-lysine  lysine cross - link was prepared by reaction of mda 
 with [ h]-lysine containing [ c]-lysine as 
 a tracer and quantified using a method similar to that described for 
 the [ h]-[c]-lysine - lactam internal standard . for quantitation , the transition between m / z 341 and m / z 306 was monitored ; it corresponds to the loss of one molecule 
 of water and one molecule of ammonia . portions of samples containing 
 the standard were then concentrated using 1 cc oasis hlb columns and 
 dried under nitrogen . the purified samples were analyzed using a thermo 
 scientific tsq vantage quadrupole mass spectrometer equipped with 
 xcalibur software for data collection and manipulation . all measured 
 mda - adduct signal in each sample was divided by the signal for the 
 [ h]-[c]-lysine  lysine cross - link standard 
 of known concentration to quantify experimental results . xpa was expressed and 
 purified as described . to modify xpa , 
 3.2 m of purified protein was incubated 
 with 500 ( 1.6 mm ) of adenine propenal for 6 h at room temperature 
 in a buffer containing 10 mm nacl and 10 mm mops ( ph 6.5 ) . a high - throughput 
 fluorescence anisotropy assay was used to measure the dna - binding 
 activity of xpa . the substrate for this assay 
 was a fluorescein - labeled y - shaped ssdna dsdna junction substrate 
 containing 8 base pairs of duplex and 12 nucleotide noncomplementary 
 ssdna overhangs at both the 3 and 5 ends . xpa was 
 modified with nhs  biotin as described previously . modified or mock - treated xpa protein and the 
 dna substrate were diluted in binding buffer ( 20 mm hepes , ph 7.9 , 
 75 m kcl , 5 mm mgcl2 , 5% glycerol , 1 mm dtt ) . fluorescence anisotropy 
 was measured using a synergy h1 plate reader ( ex = 485 nm , em = 528 nm ) . apparent dissociation constants ( kd ) were 
 determined for each individual titration by plotting fluorescence 
 anisotropy against protein concentration and fitting to a simple two - state 
 binding model with kaleidagraph ( v4.03 ) software . adenine propenal was reacted with n--acetyllysine , n--acetylhistidine , n--acetylarginine , or n--acetylcysteine 
 in 10 mm nacl buffered with 10 mm mops ( ph 6.5 ) . reaction progress 
 was monitored hourly by hplc using a c18 reverse - phase column for 
 product separation . adenine propenal eluted at 8 min and displayed 
 a maximum absorbance of 260 nm ( figures 2 and s1 ) . reaction with n--acetyllysine 
 resulted in the appearance of three new chromatographic peaks at 1.0 , 
 4.8 , and 5.3 min , which were analyzed by uv / vis , nmr , and ms ( figures 2 and s1 ) . the peak eluting 
 at 1 min displayed a uv absorbance maximum at 260 nm and an m / z of 136 , corresponding to protonated 
 adenine ( [ m + h ] ) ( 1 ) . on the basis of nmr analysis , this product 
 was identified as 3 , and lc - ms analysis gave an m / z of 243 , which corresponds to the protonated 
 species ( [ m + h ] ) . the 5.3 min peak , with a maximum absorbance 
 at 300 nm and an m / z of 413 , which 
 corresponds to the protonated species ( [ m + h ] ) , was identified 
 as 4 by nmr analysis . the total amount of product formed 
 was unaffected by variations in ph over a range of 6.5 to 9.5 . n--acetyllysine 
 ( 50 mm ) was incubated with adenine propenal ( 10 mm ) in a 50 l 
 reaction mixture for 2 h at room temperature . the column 
 eluate was monitored at 260 nm ( a ) or 300 nm ( b ) . analytes present 
 in each peak were subjected to ms , uv , and nmr spectrometry . chemical synthesis of 3 and 4 yielded 
 standards that verified the product structure assignments . the molar 
 extinction coefficients of the pure compounds (  = 33.6  
 10 m cm at 280 
 nm for 3 and  = 33.6  10 m cm at 300 nm for 4 ) were similar to that of adenine propenal (  = 32.5  
 10 m cm at 260 
 nm ) . hplc analysis of reaction mixtures of n--acetyllysine 
 and adenine propenal using diode - array detection enabled direct comparison 
 of peak areas for each compound measured at its optimal wavelength 
 ( figure 3 ) . the essentially identical absorbance 
 coefficients of 3 and 4 allowed peak areas 
 to be used to estimate relative product quantity . these comparisons 
 indicated that the major product formed from the reaction was 4 , with much lower quantities of 3 . hplc analysis 
 of product formation in the reaction of adenine propenal 
 and n--acetyllysine . the indicated amounts 
 of n--acetyllysine ( nal ) were incubated with 
 10 mm adenine propenal , and reactions were monitored hourly for 910 
 h. peaks corresponding to 3 and 4 were monitored 
 at 280 and 300 nm , respectively , by diode - array detection . variation of the amount of n--acetyllysine 
 ( from 2 to 100 mm ) in reaction mixtures while holding the amount of 
 adenine propenal ( 10 mm ) fixed demonstrated that the amount of 3 formed increased with n--acetyllysine 
 concentrations from 2 to 10 mm and then decreased somewhat at the 
 higher concentrations . in contrast , the amount of 4 produced 
 increased with n--acetyllysine concentrations 
 up to 50 mm . on the basis of the relative peak area , the ratio of 4 to 3 in most reaction mixtures was approximately 
 10:1 . estimation of the concentration of 4 using absorbance 
 at 300 nm indicated a maximum of 9 mm formed in reaction mixtures 
 containing 50100 mm n--acetyllysine 
 ( figure s2 ) . our initial hypothesis for the production of 4 was 
 that it arose by reaction of n--acetyllysine 
 with 3 , but this reaction did not occur under conditions 
 comparable to those used for the reaction of adenine propenal with n--acetyllysine . an alternative reaction scheme , 
 in which an enaminoimine cross - link between adenine propenal and n--acetyllysine is first formed and then reacts with 
 h2o to produce 3 or another equivalent of n--acetyllysine to form 4 , is shown 
 in figure 4 . if this scheme is correct , then 
 our data indicate that , at the concentrations of n--acetyllysine and adenine propenal used here , reaction of 
 the proposed intermediate with another molecule of n--acetyllysine is more favorable than its reaction with water . hplc analysis with uv or in - line nmr was unable to detect 
 the putative 
 intermediate 5 ( figure 4 ) . therefore , 
 we attempted to trap it by reduction with nacnbh3 . adenine 
 propenal was incubated with n--acetyllysine 
 in 10 mm nacl/10 mm mops ( ph 6.5 ) in the presence of nacnbh3 . we utilized lc - ms analysis of the reaction mixture to detect ions 
 with m / z 360 , which corresponds 
 to the partially reduced intermediate ( 6 ) . a peak corresponding 
 to this mass eluted at 2.9 min , consistent with the transient formation 
 of the adenine propenal adduct to n--acetyllysine 
 ( figure 5 ) . ( a ) representative lc - ms chromatogram 
 of 6 . in this 
 example , 5 mm n--acetyllysine was reacted 
 with 10 mm adenine propenal for 30 min followed by reduction with 
 50 mm nacnbh3 . ( b ) mass spectrum of the parent ion of the 
 reduced intermediate displaying m / z 360 with detection in negative ion mode . reactions of adenine propenal with n--acetylhistidine 
 and n--acetylarginine did not result in the 
 formation of new chromatographic peaks . reaction with n--acetylcysteine resulted in the formation of an oxopropenylated 
 product , which displayed m / z 216 , 
 corresponding to the deprotonated molecular ion , and its major fragmentation 
 product , m / z 86.9 . treatment of 
 the reaction mixture with nabh4 yielded the partially reduced 
 product ( m / z 218 ) and its major 
 fragmentation product ( m / z 89 ) ( figure s3 ) . to ascertain adenine propenal s ability to 
 react with protein , we incubated increasing amounts of the electrophile 
 with a fixed amount of purified hsa for 6 h , and the samples were 
 prepared for lc - ms / ms by trichloroacetic acid precipitation , resolubilization 
 of the precipitated protein pellet , reduction and carbamidomethylation 
 of cysteine residues , and proteolytic digestion with sequencing - grade 
 trypsin . the resultant proteolytic peptides were analyzed via lc - ms / ms 
 using an ltq - orbitrap velos mass spectrometer with collision - induced 
 dissociation . sequence coverage for unmodified hsa was found to be 
 90% , but exposure to increasing concentrations of adenine propenal 
 reduced coverage to a minimum value of 62% ( table 
 s1 ) . hsa peptides were interrogated for the presence of a + 54.0105 
 mass increase , as would be expected from the addition of an oxopropenyl 
 group . to verify positively identified residues , extracted ion chromatograms were 
 generated from precursor peptide masses , and theoretical masses calculated 
 from protein prospector were compared to observed masses to calculate 
 ppm mass error . for modified precursor peptides displaying a ppm mass 
 error less than 10 , ms / ms spectra were interrogated with xcalibur 
 software . at least 40% of each theoretical b- and y - product ion series was present in the observed product 
 ion spectra of positively identified peptides , and 50% sequence coverage 
 was obtained for each peptide validated . to validate the site of adduction , 
 at least two ions within a product ion series with a + 54.0105 adduction 
 mass shift were required . multiple lysine residues were found to be 
 modified in adenine propenal - exposed hsa , including lys26 , lys36 , 
 lys161 , lys183 , lys214 , lys219 , lys236 , lys249 , lys257 , lys347 , lys375 , 
 lys402 , lys413 , lys438 , lys549 , lys569 , and lys598 . residues verified 
 to have a + 54.0105 adduction mass were mapped to the crystal structure 
 of hsa ( pdb i d : 1ao6 ) and were found to be largely confined to the protein surface ( figure 6a ) . the 17 modified lysine residues represented 
 28% of the total lysines within the protein ( table 1 ) . although we determined that adenine propenal can also form 
 oxopropenylated cysteine , searching for adducts on cysteine residues 
 within hsa did not result in the identification of any modified sites . s - oxopropenal - n - acetylcysteine was stable 
 to treatment with tca as well a dtt , which are reagents used for proteomic 
 analysis , so any oxopropenylated cysteine residues on hsa - derived 
 peptides should have survived the workup prior to mass spectrometry . therefore , adenine propenal is acting as a lysine - specific 
 modifier of hsa . ( a ) modified 
 lysine residues identified in table 1 were 
 mapped to the crystal structure of hsa ( 1ao6 ) . ( b ) the most highly modified 
 residues by individual electrophiles are highlighted with the modifying 
 electrophile noted . hsa was treated at the indicated 
 molar ratios of adenine propenal and analyzed for lysine residues 
 modified with + 54.0105 . the presence of each modified lysine in a 
 sample is indicated with . the modified lysine in each peptide 
 sequence is indicated with an asterisk . equivalent lysine residues 
 within the crystal structure of hsa are provided in parentheses . the use of hsa as a biomarker 
 has primarily been described in the 
 context of electrophilic modifications at cys34 . adenine propenal did not modify cys34 , so we sought to 
 identify the most reactive lysine residue(s ) that might be used as 
 a biomarker for modification by adenine propenal . we reduced the levels 
 of adenine propenal relative to protein to a ratio of 10:1 , which 
 was the lowest ratio at which modification was observed . using the 
 interrogation methods described above to verify adduction sites , two 
 lysine residues , k190 and k351 , displayed the highest reactivity ( table 1 ) . mapping of those residues to the crystal structure 
 of hsa revealed that both are located within domain ii , a recognized 
 binding region for many compounds ( figure 6a ) . decreased hsa sequence coverage observed 
 with increasing concentrations of adenine propenal suggested that 
 adducts impede protein digestion and/or fragmentation required for 
 peptide identification . lysine cross - links are formed 
 from the reaction of adenine propenal with n--acetyllysine , 
 we hypothesized that at high concentrations of adenine propenal , lysine lysine 
 cross - links may be induced in hsa . to investigate the presence of 
 cross - links , we interrogated previously acquired proteomics data for 
 a + 36 adduct mass , corresponding to an unreduced 3-carbon linkage 
 between lysine residues . lysine cross - links , 
 which was not surprising considering the decreased sequence coverage 
 and potential hindrance of peptide fragmentation by cross - links . although 
 cross - linked sites were not identified with these algorithm - based 
 data searching methods , we manually interrogated the data to look 
 for the presence of precursor peptides containing cross - linked residues . to predict lysine residues likely to participate in cross - link formation , 
 we performed in silico analysis of the hsa crystal 
 structure ( 1ao6 ) . carbon bond is approximately 1.5  
 in length , we hypothesized that the 3-carbon cross - link would be 57 
  long . flexible , dynamic protein movements in solution may result 
 in cross - link formation between lysines at a distance greater than 
 7  . we therefore examined the crystal structure to identify 
 lysine residue pairs separated by a distance of less than or equal 
 to 20  as potential sites of cross - link formation . following 
 identification of these residues , tryptic peptide sequences were determined 
 from the primary sequence , and theoretical peptide masses containing 
 the cross - link mass of + 36 were calculated using protein prospector . we then thoroughly interrogated the data manually for the presence 
 of ions of the theoretical precursor peptide masses . no evidence for 
 the presence of cross - link - containing peptides was obtained . as proteomics - based mass spectrometric analyses were unsuccessful , we employed a different analytical approach to search for lysine  lysine 
 cross - links , in which modified hsa was degraded to the level of amino 
 acids , and the resultant hydrolysate was analyzed by ms . this methodology 
 employed selected reaction monitoring to identify analytes exhibiting 
 a mass transition from m / z 329 to m / z 294 , as exhibited by analysis of the 
 lysine  lysine cross - link standard . addition of a [ h]-[c]-4 internal standard ( m / z 341 to m / z 306 
 transition ) enabled identification of analyte - containing fractions 
 during purification by scintillation counting and quantification by 
 stable isotope dilution . application of this approach to hsa that 
 had been incubated with adenine propenal resulted in the identification 
 of 30  12 ng cross - link / mg protein in vehicle - treated hsa and 
 57  17 ng cross - link / mg protein in adenine propenal - treated 
 hsa . these differences were not statistically significant , supporting 
 the conclusion that adenine propenal did not induce intramolecular 
 cross - links in hsa . having confirmed 
 adenine propenal s capacity to modify lysine residues in hsa , 
 we investigated its reaction with xpa . adenine propenal was incubated 
 with xpa for 6 h at room temperature , and the samples were prepared 
 for and analyzed by lc - ms / ms using the same methods as described for 
 hsa . as for hsa , peptides were interrogated for the presence of a 
 + 54.0105 mass addition , and rigorous criteria were applied to peptide 
 and site of adduction identification . of note , three 
 of these , k145 , k183 , and k204 , are located in the proposed dna - binding 
 domain of xpa ( figure 7a ) , and two , k63 and k67 , are known sites of acetylation that reduces 
 xpa s interaction with replication protein a. adenine propenal modifies xpa and reduces its dna - binding activity . 
 ( a ) lysine residues modified with an adduct mass of + 54.0105 were 
 mapped to the xpa nmr solution structure ( 1d4u ) . ( b ) plots of the change in fluorescence 
 anisotropy for a y - shaped 8/12 ssdna  dsdna junction substrate 
 ( 50 nm ) versus added protein treated with dmso , adenine propenal , 
 or nhs - biotin . purified xpa was incubated with 
 a 500-fold molar excess of adenine propenal and analyzed for lysine 
 residues modified with + 54.0105 . modification of residues within the xpa dna - binding 
 domain has 
 the potential to affect dna - binding activity . using a plate - based 
 fluoresence anisotropy assay , the effect of adenine propenal - mediated 
 oxopropenylation on xpa s affinity for a y - shaped ssdna fitting 
 of the data for binding of unmodified xpa to the fluorescein - labeled 
 20-nucleotide substrate provided a dissociation constant ( kd ) of 300  30 nm ( figure s4 ) . binding affinities for the 20-nucleotide substrate 
 were measured for purified xpa incubated with vehicle ( dmso ) , adenine 
 propenal , or nhs  biotin ( biotin n - hydroxysuccinimide 
 ester ) ( table 3 ) . as expected , incubation of xpa with dmso had no effect on its dna - binding 
 activity . in contrast , exposure to nhs  biotin , which efficiently 
 modifies lysine residues and disrupts the dna - binding activity of 
 many proteins , resulted in a nearly complete loss of xpa s dna - binding affinity . xpa modified with adenine propenal bound to the substrate , but with 
 a greater than 3-fold reduction in affinity as compared to that of 
 the unmodified protein ( figure 7b ) . in fact , 
 oxopropenylation of the protein reduced the affinity to such an extent 
 that it could not be accurately measured ; thus , the kd value reported in table 3 ( 1 
  0.1 m ) is an estimate . dissociation constants ( kd ) were determined by fitting fluorescence anisotropy 
 data to a simple two - state binding model . the biotin - modification of xpa causes such a large loss of dna - binding 
 affinity that there is no significant change in fluorescence anisotropy ; 
 hence , a kd value could not be determined . covalent 
 modifications of dna and proteins by electrophiles produced 
 during oxidative stress have multiple consequences on cellular function , 
 including mutagenesis , stress responses , cellular dysfunction , and 
 ultimately cell death . all of these may contribute to pathological 
 processes such as carcinogenesis , neurodegeneration , atherosclerosis , 
 and chronic inflammation . however , elucidation of the exact mechanisms 
 by which specific macromolecular damage leads to pathology requires 
 a better understanding of the sites and nature of that damage . the most frequently used biomarker of electrophile damage to protein 
 during oxidative stress is the change in global protein carbonylation . although useful as a general indicator that 
 stress has occurred , this approach does not identify the exact type 
 of modification or the modifying electrophile . recent developments 
 in highly sensitive ms - based proteomics techniques have allowed for 
 expansion of this approach to the investigation of the specific types 
 of modifications induced and the impact of these modifications on 
 protein activity . the resultant increased ability to identify specific 
 protein modifications induced by a single electrophile enables the 
 search for biomarkers that distinguish between different mechanisms 
 of oxidant damage . the generation of adenine propenal occurs 
 predominantly in intact 
 dna , suggesting that most of its targets will likely be dna - associated 
 proteins in the nucleus or mitochondria . nevertheless , we initiated 
 our investigation of adenine propenal s protein - modifying profile , 
 using hsa as the model protein . hsa s reactivity with electrophilic 
 agents has been studied extensively in the past , allowing us to place adenine propenal - dependent modifications in 
 the context of those existing data . for example , teucrin a was found 
 to preferentially modify lys351 and lys545 , while 4-hydroxynonenal 
 modifies lys195 , lys199 , lys233 , and lys525 , and polycyclic aromatic 
 hydrocarbon epoxides modify lys137 . mda has been shown to 
 modify multiple lysine residues including lys136 , lys174 , lys240 , 
 lys281 , lys525 , and lys541 . as mda and 
 adenine propenal both produce oxopropenyl adducts on lysine residues , 
 it is interesting to note that the major sites of adduction for the 
 two electrophiles are different : lys190 and lys351 for adenine propenal 
 versus lys525 for mda . these findings suggest that distinct hsa modification 
 patterns may be used as markers for different electrophiles ( figure 6b ) . clearly , the use of hsa as a biomarker of electrophile 
 damage is attractive , given its high concentration in the blood and 
 ease of sampling . however , as noted above , adenine propenal generation 
 is largely confined to intact dna , so it remains to be seen if enough 
 of the electrophile reaches the circulation to allow the modification 
 of hsa to serve as a biomarker of exposure . the impact of electrophiles 
 has long been studied in the context 
 of covalent modification of dna and its potential to lead directly 
 to mutations . however , adenine propenal produced in the nucleus also 
 has the potential to modify dna - associated proteins , including those 
 involved in dna repair . we report here that adenine propenal covalently 
 modifies the ner protein xpa in vitro and that this 
 modification leads to a reduction in xpa s dna - binding activity . oxopropenylation of residues known to be acetylated further suggests 
 that adenine propenal - dependent modification could result in failure 
 of regulation of xpa s activity through sirt-1-dependent deacetylation . xpa is a critical component of the ner machinery , 
 without which repair activity ceases . mutations in xpa , particularly in the dna - binding domain , are associated 
 with severe xp clinical phenotypes . thus , 
 it is possible that decreased xpa - binding activity resulting from 
 adenine propenal - dependent modification could result in reduced ner 
 activity in cells and increased potential for mutagenesis and carcinogenesis . it should be noted that , in this model , the modification of repair 
 proteins may have a synergistic effect on mutagenesis by preventing 
 the repair of the original dna lesion . in this case , even moderate 
 reductions in the activity of the repair proteins have the potential 
 to result in significant increases in genomic instability . further 
 analysis will be required to determine whether reactive base propenal 
 species modify dna repair proteins in cells under oxidative stress 
 and to characterize the effects of modification on dna repair activity in vivo .