characterization of n-terminal formaldehyde adducts to hemoglobin

Research Article

Received: 17 September 2010 Revised: 6 January 2011 Accepted: 22 January 2011 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2011, 25, 1043–1050

Characterization of N‐terminal formaldehyde adductsto hemoglobin†

Maria Ospina*, Alina Costin, Adrienne K. Barry and Hubert W. VesperDivision of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta,GA 30341, USA

A procedure to prepare and purify adducts of formaldehyde (FA) to the N‐terminus of peptides was developed.FA‐VHLTPEEK and FA‐VLSPADK were produced with purities >95% upon incubation of the peptides with FA inphosphate‐buffered saline (PBS) at a pH level of 7.4. The peptides were purified by preparative liquidchromatography and were characterized by their retention times in liquid chromatography, their fragmentationpatterns obtained by tandem mass spectrometry, and their accurate mass and nuclear magnetic resonancemeasurements. This is the first time an imidazolidone‐type structure has been reported for FA adducts. The samepeptides were identified in tryptic digests of human hemoglobin incubated with FA at physiological conditions andin human hemoglobin specimens. These peptides are suitable for use as calibrators for the quantitative assessmentof internal exposure to FA. Published in 2011 by John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.4954

Formaldehyde (FA) is among the 25 chemicalswith the highestproduction in the United States.[1] It is used as a preservative[2]

and sterilizer[3] and is found in various products such as dyes,textiles, plastics, paper products, cosmetics, and householdcleaners. The International Agency for Research in Cancer(IARC) Working Group, which reviewed FA studies, con-cluded that FA exposure causes nasopharyngeal cancer andclassified FA as carcinogenic to humans (group 1).[4] TheNational Toxicology Program classifies FA as “reasonablyanticipated to be a carcinogen in humans”,[1] and the U.S.Environmental Protection Agency considers FA a probablehuman carcinogen (group B1).[5]

People are exposed to FA through vapors emitted fromproducts containing this chemical, such as resins, glues,buildingmaterials, and furniture. Approximately 90% of thesevapors are readily absorbed by the the respiratory tract.[6]

Other major sources of FA exposure are cigarette smoke[7,8]

and endogenous production during normal cellular metabo-lism through enzymatic or nonenzymatic reactions, such asthe metabolism of serine, glycine, and choline.[9] FA is also adetoxification product of demethylation ofN,O‐ and S‐methylxenobiotics during cellular metabolism.[10,11]

Formaldehyde is rapidly metabolized to formate by severalenzymatic reactions and little, if any, free FA can be found inthe blood of humans or animals exposed to FA.[12] Theendogenous concentrations of FA in human blood are about2–3μg/g of blood and are similar to concentrations measuredin the blood of monkeys and rats.[6,13] FA is highly reactive

* Correspondence to: M. Ospina, Division of LaboratorySciences, National Center for Environmental Health,Centers for Disease Control and Prevention, Atlanta, GA30341, USA.E-mail: [email protected]

† This article is a U.S. Government work and is in the publicdomain in the U.S.A.

Rapid Commun. Mass Spectrom. 2011, 25, 1043–1050

towards biomolecules and can react with side chains ofarginine, cysteine, histidine, lysine, and N‐terminal aminoacids of proteins. These reactions change the biologicalfunctions of the proteins.[14] The in vitro formation of theseadducts is mostly affected by the FA concentration, pH level,and temperature during the first hours of incubation.[15]

Hemoglobin adducts, especially adducts at the N‐terminus,have been successfully used as biomarkers of exposureto reactive chemicals such as acrylamide, butadiene, andethylene oxide, among others.[16–19] Hemoglobin adductsreflect the exposure accumulated over the last 120 days,making them good biomarkers for long‐term expo-sures.[16,20,21] Some researchers have shown the use of N‐terminal peptide adducts of hemoglobin to measure exposureto 1,3‐butadiene in non‐smoking volunteers,[22] and in mouseand rats.[23,24]

The reaction products of hemoglobin with sugars andcertain aldehydes have been studied,[15,25–30] and the struc-tures of adducts such as Schiff bases and imidazolidinones aredescribed in the literature.[31–33] However, little is knownabout the reaction products of FA at the N‐terminus ofhemoglobin.[29,34]

In this study, we investigated theN‐terminal, FA adducts byusing VHLTPEEK and VLSPADK as model peptides. Thesepeptides are obtained from trypsin digestion of hemoglobinand represent the N‐terminal peptides of the beta and alphachains of hemoglobin, respectively, and are potential biomar-kers of FA exposure.

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EXPERIMENTAL

Chemicals

VHLTPE, VHLTPEEK, and VLSPADK, which are peptideswith the same N‐terminal sequence as beta and alpha chainsof hemoglobin, were synthesized by Bachem (King of

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Prussia, PA, USA). The purities of these peptides were higherthan 95% on the basis of high‐performance liquid chromatog-raphy (HPLC) analysis. American Chemical Society (ACS)grade FA solution, 37% weight by weight, and formicacid were obtained from Acros Organics (Geel, Belgium).Trifluoroacetic acid in ampules was purchased from PierceChemical Co. (Rockford, IL, USA). Trypsin Gold, massspectrometry grade, was obtained from Promega (Madison,WI, USA). Acetonitrile and water, both OPTIMA liquidchromatography/mass spectrometry (LC/MS) grade, wereobtained from Fisher Chemical Company (Itasca, IL, USA).Ferrous hemoglobin Ao (HbAo) and all other chemicalsand reagents were purchased from Sigma Chemical Co.(St. Louis, MO, USA).

Preparation and purification of FA‐peptide adducts

VHLTPEEK and VLSPADK were incubated with a 100‐mMsolution of FA at 1:1, 1:10, and 1:20 peptide‐to‐FA molarratios for 4 days in 200‐mM PBS buffer with a pH level of 7.4at 37 °C to determine the optimum peptide‐to‐FA ratio forformation of FA‐peptide adducts. A ratio of 1:20 gave thehighest yield.FA‐peptide adducts were purified by preparative liquid

chromatography (Prep LC) with an Agilent 1100 LC system(Agilent Technologies, Santa Clara, CA, USA) equippedwith a quaternary pump, a photodiode array (PDA) detector,and a FC 204 fraction collector (Gilson, Inc., Middleton, WI,USA). Separation was obtained at room temperature with aJupiter 4μm Proteo 90Å column (50× 4.6mm; Phenomenex,Torrance, CA, USA). The mobile phase was water with0.025% trifluoroacetic acid (TFA) (A) and acetonitrile with0.025% TFA (B). Separation was achieved at 1mL/min with alinear gradient of 5%–17% B. After separation, the columnwas washed at 60% B for 2min and equilibrated at 5% B for8min. The UV absorbance at 210 nm was monitored, andfractions were collected every 30 s (0.5mL per fraction). Eachfraction was tested for the presence and purity of thepeptides of interest by LC/MS as described below. Fractionscontaining peptide with the FA adduct were pooled, frozen,and lyophilized in a SavantMicroModulio freeze dryer (SavantInstruments, Holbrook, NY, USA). Lyophilized FA‐peptideadducts were stored at −70 °C until use.

LC/MS conditions for testing prep LC fractions(short method)

Peptide separation was performed at 30 °C with a Jupiter 4μmProteo 90Å column (50.0× 2.0mm; Phenomenex, Torrance, CA,USA) at a flow rate of 200 μL/min determined by a SurveyorHPLC system (ThermoElectron, San Jose, CA, USA). Themobile phase was water with 0.025% TFA (A) and acetonitrilewith 0.025% TFA (B). Separation was achieved with a lineargradient of 5%–30% B. The column was then washed at 60% Bfor 4min and equilibrated at 5% B for 6min. The samples werekept at 8 °C in the autosampler tray, and 10μL of each fractionsolutionwere injected. The LCsystemwas connected to anLCQDeca ion trap mass spectrometer (ThermoFinnigan, San Jose,CA, USA). Samples were analyzed by electrospray ionization(ESI) in the positive ionmode. The ESI voltagewas set to 4.5 kV,the sheath and auxiliary gases to 80 and 20 arbitrary units,respectively, and the heated capillary temperature to 250 °C.

wileyonlinelibrary.com/journal/rcm Published in 2011 by John Wi

Data in the ion trap were obtained from 150–2000Da with 3micro scans and 200ms maximum injection time.

Assessment of purified FA‐peptide adducts

The purified and lyophilized FA‐peptide adducts were redis-solved in 0.1% formic acid, and LC/MS/MS analysis wasperformed in an LTQOrbitrap Velos hybrid mass spectrometer(Thermo Scientific, Bremen, Germany) equipped with a HESI‐2ion source interface. Column and chromatographic conditionswere the same as those in the previously described shortmethod except that the mobile phase was water with 0.1%formic acid (A) and acetonitrile with 0.1% formic acid (B). Themass spectrometer was set up to obtain full scan MS spectra(scan range: 300–2000) at a resolution of 60 000 (FWHM) withpredictive ion injection time. Full scan MS/MS spectra in thelinear ion trap were obtained by collision‐induced dissociation(CID) at a normalized collision energy of 35% as the activationmode. An isolationwidth of 2Da, an activation q of 0.25, and anactivation time of 30ms were used for selecting the precursorion. Higher energy collision‐induced dissociation (HCD) ata normalized collision energy of 40% was also used to obtainfull scan MS/MS data without the limitations of the low masscut‐off of the linear ion trap.

FA‐peptide incubation reaction time course

Using the incubation conditions producing the highest yieldsfor the peptide adducts, we assessed the time course of thereaction. Samples consisting of 750μLof the incubated solutions(1:20 molar ratio for VHLTPEEK and 1:30 molar ratio forVLSPADK) were incubated at 37 °C in the HPLC autosampler,and 5μL of reaction solution were injected and analyzed everyhour by LC/MS (short method) described previously.

Preparation of FA‐hemoglobin adducts

HbAo (100mg) was dissolved in 5mL of 200mM PBS buffer.The hemoglobin content was measured with a Hemocueinstrument (Hemocue, Lake Forest, CA, USA). Aliquots ofhemoglobin solution (500μL) were mixed with FA at 1:3,1:30, and 1:100 molar ratios of hemoglobin to FA (HbAo‐FAsolution). The total volume was adjusted to 750μL with200mM PBS buffer, and samples were incubated for 4 days at37 °C. Half of the sample was kept for further enzymaticdigestion, and the other half was used for globin precipita-tion. Globin was isolated by using the procedure describedby Mowrer et al.[35] In brief, globin was precipitated byadding 6 volumes of a cold 50mM solution of hydrochloricacid in isopropanol followed by centrifugation for 45min at3000 g and 4 °C. The supernatant containing the globin waswashed three times with cold ethyl acetate to precipitateglobin. The precipitate was washed with pentane and left todry overnight in the chemical fume hood. The globin wasstored at −70 °C until further use.

Digestion of FA‐incubated proteins

Approximately 2mg of HbAo‐FA solutions (130μL) weredissolved in 1mL of water; 250μL of this solution (about500μg HbAo) and 25μL of internal standard (VHLTPEpeptide solution, 0.25μg/μL in water) were combined in amicrocentrifuge tube and mixed with 250μL of 100mMammonium bicarbonate with a pH level of 8.5. Globin and

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N‐terminal formaldehyde adducts to hemoglobin

hemoglobin were digested with trypsin at a 1:25 ratio ofenzyme‐to‐protein for 18 h at 37 °C. Digestion was stoppedby adding 10μL of formic acid.

Analysis of tryptic digests

Peptide separation was performed at 30 °Cwith a Jupiter 4μmProteo 90Å column (50 × 2.0mm; Phenomenex, Torrance, CA,USA) at 250 μL/min. Themobile phasewaswaterwith 0.025%TFA (A) and acetonitrile with 0.025% TFA (B). Separationwas achieved with a linear gradient of 5%–60% B. The columnwas then washed at 60% B for 4min and equilibrated at 5% Bfor 10min. The samples were kept at 8 °C in the autosamplertray, and 10μLwere injected. The LC systemwas connected toan LCQ Deca ion trap mass spectrometer (ThermoFinnigan,San Jose, CA, USA). Samples were analyzed by ESI in thepositive ion mode. Area ratios of peptides to an internalstandard (VHLTPE) were used to compare the formation ofFA adducts (FA‐VHLTPEEK and FA‐VLSPADK) at differentprotein‐to‐FA ratios.

Nuclear magnetic resonance (NMR) spectroscopy

The peptide‐containing FA‐adductswere characterized by usingthe following NMR techniques: proton, carbon‐13, correlationspectroscopy (COSY), total correlation spectroscopy (TOCSY),distortionless enhancement by polarized transfer (DEPT),heteronuclear multiple quantum coherence (HMQC), andheteronuclear multiple‐bond correlation (HMBC). These techni-ques were performed at room temperature on INOVA 600 andUNITY Plus 600 instruments (Varian, Inc., Palo Alto, CA, USA)at EmoryUniversity, NMRResearch Center (Atlanta, GA, USA).Peptides were dissolved in deuterated dimethyl sulfoxide. Allchemical shifts are reported relative to tetramethylsilane.

Digestion of red blood cell samples and identificationof peptides

Whole blood was obtained from a blood bank and thecorresponding red blood cells (RBC) were isolated. Samples(1mg) were digested with trypsin as described above. Thedigested samples were diluted 1:1 with 0.1% formic acid and10μL was analyzed by LC/MS with an LTQ Orbitrap Velosmass spectrometer equipped with a HESI II ion source(Thermo Scientific). The mass analysis consisted of two scans,one full FT scan from 350–500Da at 60000 resolution followedby a data‐dependent scan in the trap; this scan was triggeredby a mass list containing the m/z values of the doublycharged ions of interest. Isolation widths of 2Da and CID atnormalized collision energies of 35% were used. The samesample was analyzed also by HCD with an isolation width of2Da and normalized collision energy of 40% to see fragmentions in the low‐mass range. Retention times and MS/MSspectra from the digested sample were compared to those ofthe purified peptides.

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RESULTS AND DISCUSSION

FA is known to react with proteins. The goal of this studywas to investigate the formation of FA‐hemoglobin adductsat the N‐terminus of the protein chains and to characterizethe adducts formed.

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Formation and identification of peptide adducts

The peptides used for the reaction with FA were firstanalyzed by using LC/MS with an LCQ mass spectrometer.VHLTPEEK and VLSPADK incubated with FA presentedsignals for singly charged ions at m/z 964.5 and 741.4,respectively, that were 12Da higher than those of theunmodified peptides. An increase of 12Da for both peptidesagrees with the incorporation of one FA equivalent. Thecorresponding doubly charged ions were also present at m/z482.8 and 365.2 for FA‐VHLTPEEK and FA‐VLSPADK,respectively.

Accurate‐mass high‐resolutionmeasurements performed ina ThermoLTQOrbitrapVelosmass spectrometer at a resolvingpower of 60 000 (FWHM) were better than 2.5 ppm in the full‐scanmode for FA‐VLSPADK, despite the theoretical values forthe [M+H]+ and [M+ 2H]2+ ions for FA‐VLSPADK,which arem/z 741.4147 and 371.2112, respectively. For FA‐VHLTPEEK,accurate mass measurements errors were below 4ppmfrom the theoretical values of m/z 964.5104 and 482.7591 forthe [M + H]+ and [M + 2H]2+ ions, respectively.

More than 90% of the peptides were converted into themono‐FA‐peptide adducts when incubated with FA at a 1:20ratio (Fig. 1). The higher retention times of the FA peptides areconsistent with the higher hydrophobicities of the new species.Under the conditions of the experiment, only VHLTPEEKshowed a double FA adduct, which was well separated bychromatography (11.6min). A higher FA‐to‐peptidemolar ratiowas needed for the reaction with VLSPADK (1:30) to producethe same adduct yield as the reaction with VHLTPEEK. Thepurity of the FA peptides was higher than 95% as determinedby HPLC with UV‐detection. The peptide standards appearstable in digestion buffer solution at room temperature for atleast 1week and at 37 °C, 48 °C and 53 °C for at least 48h. Thestandards are stable for at last 2 years at −70 °C.

CID and HCD of the doubly charged ions of FA‐VHLTPEEK and FA‐VLSPADK (Figs. 2 and 3) performedon the LTQ Orbitrap Velos instrument provided furtherinformation on the location of the FA‐derived group withinthe peptides. For both compounds, the CID spectra pre-dominantly yielded modified b‐type ions (b*) and y‐typeions.[36] An abundant doubly charged ion, despite the loss ofwater, is observed for both compounds in CID. The HCDspectrum provides more information about low m/z frag-ments that are usually excluded from the CID spectra due tothe low mass cut‐off of CID with resonance excitation.

HCD of FA‐VHLTPEEK (Fig. 2) provides confirmation ofthe amino acid composition of the peptides, as severalimmonium ions are observed. Ions at m/z 102, 110, and 129correspond to the immonium ions of glutamic acid, histidine,and lysine, respectively. The fragment at m/z 84 correspondsto the valine immonium ion plus 12Da (V*). Likewise, the ionat m/z 122 corresponds to the histidine immonium ion plus12Da (H*). The occurrence of both V* and H* indicates thatthe FA adduct is attached to both amino acid residuesforming a cyclic structure. For FA‐VLSPADK (Fig. 3),cleavage at the proline residue yields an abundant y5 ion atm/z 517. The b2* and b3* ions at m/z 225 and 312 correspondto the alkylated fragments FA‐VL and FA‐VLS, respectively.The HCD spectrum of FA‐VLSPADK also shows immoniumions for valine, leucine, and lysine at m/z 72, 86, and 129. Ionsat m/z 84 and 98 corresponding to the immonium ions of

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Figure 2. MS/MS spectra of the [M + 2H]2+ ion of formaldehyde (FA)‐VHLTPEEK (precursor ion at m/z 482.8). * indicatesfragments containing the modification.

Figure 1. Effect of formaldehyde (FA) concentration on peptide adduct formation. Total ion chromatogram (TIC) show thepeaks corresponding to the control peptides (VLSPADK and VHLTPEEK, retention times 6.8 and 7.8min, respectively) and theformation of formaldehyde adducts (FA‐VLSPADK and FA‐VHLTPEEK, retention times 9.5 and 8.7min, respectively) as afunction of the amount of formaldehyde used.

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Figure 3. MS/MS spectra of [M + 2H]2+ of formaldehyde (FA)‐VLSPADK (precursor ion at m/z 371.2). * indicates fragmentscontaining the modification.

Figure 4. Suggested reaction scheme for the formation offormaldehyde adducts on peptides.


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valine and leucine plus 12Da are identified in the HCDspectrum as V* and L*, respectively. These findings indicatethat the FA‐adduct is located at the N‐terminal of valine andthat a cyclic structure is involved.MS/MS data did not show any signals corresponding to

modification on the lysine. The peptides were incubated in PBSbuffer at pH level 7.4 to simulate physiological conditions.These conditions are well below the pKa of lysine of 10.53.Therefore, the amino group is mainly protonated, which mayexplain the inhibition of formation of adducts on this aminoacid. This finding agrees with previous reports on isoprene.[18]

Figure 4 shows a proposed scheme for the reaction of FAon the N‐terminal valine of the peptides. The literature hasreported that reaction of hemoglobin with acetaldehyde canresult in two types of adducts of the same mass, one being aSchiff base (imine) and the other a 2‐methyl‐imidazolidin‐4‐one.[31] FA is expected to react similarly. Sodium borohydridecan efficiently reduce imines into secondary or tertiaryamines.[37] The FA peptides were reacted with NaCNBH3, areducing agent, to test for the presence of the imine. Theappearance in the LC/MS spectrum of a peak 2Da higher thanthe original peptide was expected but not observed. Conse-quently, the imine structure was considered absent for the FApeptide, an assumption confirmed by NMR analysis. Toconfirm the position of the FA adduct at the N‐terminal valineand the presence of the imidazolidone group, NMR spectrawere obtained for the native and the modified peptides. The

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proton NMR spectrum confirmed the imidazolidone structureresulting from FA reaction on the valine. The two methylgroups of valine in VHLTPEEK are equivalent, showingonly one doublet at 0.91 ppm because of free rotation. InFA‐VHLTPEEK, these two methyl groups are not equivalent;

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they split into two doublets, one doublet shows at 0.72 ppmand the other at 0.88 ppm, indicating restriction of the rotationdue to the imidazolidone ring. More direct proof of theformation of the imidazolidone ring in proton NMR is thedisappearance of proton peaks of the NH2 of valine andNH ofadjacent histidine. These two proton signals in VHLTPEEK areat 8.11 and 8.76 ppm, respectively. Also, two additional peaksat 4.53 and 4.63 ppm appeared in the spectrum of FA‐VHLTPEEK and integrated for one proton each in the protonNMR spectrum. The COSY and TOCSY spectra show thatthese two protons are coupled to each other. The HMQCspectrum shows that these two protons are correlated to onecarbon at 57.75 ppm, indicating that it is a CH2 group. There isalso an indirect proof of the ring formation. In the COSYspectrum of FA‐VHLTPEEK, the coupling of the α‐H (at3.64 ppm) and the β‐H (at 1.96 ppm) of the modified valine isnot observed, while in the COSY spectrum of the VHLTPEEK,this coupling is very clear. The ring formation constrains therotation of bonds and the dihedron angle of α‐CH and β‐CHbonds is close to 90°. All protons having a 90° dihedron anglewith each other will not show their couplings. All other protonpeaks show up at similar chemical shifts in the native andthe adducted peptides. Consistent results were obtained forFA‐VLSPADK. The formation of an imidazolidone structurefrom FA‐N‐terminal peptide adducts of hemoglobin agreeswith findings from other researchers on reactions of insulinwith FA[38] and of hemoglobin with acetaldehyde.[31] Thisstudy is the first report of the imidazolidone structure for FAand these peptides.

Time course of the reactions between VLSPADK andVHLTPEEK with FA

The purpose of this experiment was to monitor theincubation of peptides with FA over time and obtaininformation about the time needed to reach equilibrium ofthe reaction and about the reactivity of the alpha and betachain peptides. The concentration of the adducted peptidespeaked at about 13 h for the formation of FA‐VLSPADK, andthe formation of FA‐VHLTPEEK peaked at around 7.5 h. Thenatural logarithm of the concentration of the native peptidewas plotted against incubation time in seconds, and a linearrelationship was observed, indicating a first‐order reactionfor both native peptides. From these equations, the calculatedhalf‐life of the VLSPADK reaction is 5.1 h and for VHLTPEEKis 4.7 h. These reactions were performed with a 1:30 molarratio for VLSPADK to FA and a 1:20 molar ratio forVHLTPEEK to FA. This experiment showed that the reactionof FA with VLSPADK is slower than the reaction withVHLTPEEK (Fig. 5). To achieve the same yield under similarconditions, more FA was needed for the reaction withVLSPADK. In our experiment, this reaction was achievedby using a 1:30 molar ratio of VLSPADK to FA. Given theconditions of the reaction (pH7.4), thenearbyhistidine (pKa 6.0)in the VHLTPEEK acts as a base that can abstract the proton inthe protonated imine formed upon reaction with FA.Histidine, a cyclic amino acid, can better accommodate thecharge by resonance and eventually loses the charge withthe buffer and returns to its original state. In general, theneighboring histidine increases the electronegativity of thenitrogen in valine, making it a much stronger nucleophile andfavoring the formation of the imidazolidone.


Identification of FA‐VLSPADK and FA‐VHLTPEEK intryptic digestions of HbAo incubated with FA

Trypsin digestion of hemoglobin and globin incubated withFA was performed to identify FA‐modified peptides formedduring the incubation procedure. Mass spectrometric analy-sis of this digestion detected the same FA‐VHLTPEEK andFA‐VLSPADK peptides that were formed by incubation ofthe native peptides with FA (Fig. 6). Retention times, a fullscan, and MS/MS spectra confirmed the identity of thesepeptides. An internal standard was added before digestion,and the area ratio of FA peptides to the internal standardpeptide was monitored. An increase in the area of theadducted peptide generated during trypsin digestion wasobserved as the amount of FA added to the reaction withhemoglobin was increased (Table 1). This increased area ofadducted peptide was also accompanied by a decrease in thearea ratio of the corresponding unmodified peptide to theinternal standard. This dose response indicates that thesepeptides might be suitable as biomarkers of exposure. Datareported for the reaction of acrylamide with hemoglobin[39]

show a similar response. Further experiments conducted atrelevant FA exposure concentrations are needed.

Analysis of human blood samples

FA‐VLSPADK and FA‐VHLTPEEK were also identified inhuman RBC samples upon digestion of 1mg of Hb (50μL ofRBCs with hemoglobin concentration of 2 g/dL) and analysisof the digested samples. The MS/MS spectra using CID andHCD as activation methods as well as the retention times ofthe signals observed in the RBCs digests matched well thoseof the peptides incubated with FA and the peptides obtainedfrom the digestion of the hemoglobin incubated with FA.Analysis of the same erythrocyte sample stored at −70 °C forover 2 years does not indicate any degradation of theanalytes. These findings indicate that these adducts can bevaluable biomarkers of formaldehyde exposure. However,further studies are warranted to assess the in vivo lifetime andformation of these adducts in response to formaldehydeexposure. The study of hemoglobin adducts, in general, doesnot allow for distinguishing between endogenous vs.exogenous exposure sources. They do reflect overall internalexposure that could lead to health effects. Thus, measuringthese potential biomarkers may provide valuable informationabout health effects associated with FA exposure. They canalso be used to identify people or groups with unusually highinternal exposures by comparing individual levels with thosecommonly observed in the population, to assess trends inexposure overtime, and to perform other assessmentscommonly done in human biomonitoring studies.

CONCLUSIONS

We report for the first time the formation of imidazolidonesat the N‐terminus of the alpha and beta chains of hemoglobinupon reaction of hemoglobin with FA. We were able toprepare adducts of FA on the N‐terminal peptides ofhemoglobin with purities higher than 95%. The modificationoccurred at the N‐terminal valine, and an imidazolidone ringstructure fits the data obtained for these compounds well. Weidentified the same FA adducts in trypsin digests of


Figure 5. Relationship between peptide concentration and reaction time for the incubation of peptides with FA in PBS buffer(pH 7.4) at 37 °C.

Figure 6. Tryptic digest of HBAo incubated with FA 1:3. Top trace shows total ion chromatogram (TIC). Bottom tracescorrespond to extracted ion chromatograms for the singly charged ions of internal standard (VHLTPE), VHLTPEEK,FA‐VHLTPEEK, VLSPADK and FA‐VLSPADK, respectively. Retention times are indicated for each compound.

Table 1. Area ratio of formaldehyde (FA) peptides to internal standard (VHLTPE) forhemoglobin (HbAo) and globin incubated at different protein‐to‐FA molar ratios

Sample FA‐VLSPADK/IS FA‐VHLTPEEK/IS

HbAo‐FA 1:3 0.166 0.097HbAo‐FA 1:100 0.481 0.466Globin(HbAo‐FA 1:3) 0.01 0.023Globin(HbAo‐FA 1:3) 0.032 0.094


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hemoglobin incubated with FA, and we showed a doseresponsewith increasing amounts of FA. The same FA‐peptideadducts were identified in a tryptic digestion of RBCs. TheseFA adducts appear suitable as potential biomarkers ofexposure to FA. Also, FA‐VLSPADK and FA‐VHLTPEEK canbe used as calibrators in our method for assessing exposure toFA in human blood samples.

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AcknowledgementsSpecial thanks are extended to Drs. Shiaoxiang Wu and BingWang from the NMR Research Center at Emory Universityfor assistance with NMR data interpretation.

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DISCLAIMER

The findings and conclusions in this report are those of theauthors and do not necessarily represent the official positionof the Centers for Disease Control and Prevention.

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characterization of n-terminal formaldehyde adducts to hemoglobin

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