New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I

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(1)Original article. Maria Pieri1,2 Nadia Miraglia2 Giuliana Genovese2 Rossella Guadagni2 Antonio Acampora1 Nicola Sannolo2. 1Department. from the 25:1 digestions allowed the determination of internal secondary proteolytic sites, not reported in literature. In both cases, a non-negligible proteolysis was observed only when a reaction time of 18 hrs was used. Discussion: results showed a progressive enrichment of modified hemoglobin with respect to the unmodified one, particularly for the α-chain and represent a promising initial step in the development of a selective purification procedure to be applied to protein adducts analysis. KEY WORDS: Biomarkers, Calpain, Haemoglobin, Mass Spectrometry.. In te r. of Advance Biomedical Science – Forensic Medicine, Histology and Anatomy Section, University of Naples “Federico II”, Naples, Italy 2Department of Experimental Medicine – Section of Hygiene, Occupational and Forensic Medicine – Occupational Medicine Area, Second University of Naples, Naples, Italy. na zi on. al i. New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I. Introduction. iz. Abstract. IC. Ed. Background: hemoglobin adducts are considered suitable biomarkers to evaluate human exposure to electrophile agents. One of the procedures used for the molecular dosimetry of hemoglobin adducts is the enzymatic digestion of the protein and the subsequent quantification of modified peptides by mass spectrometry. The aim of the study is to evaluate the ability of Calpain to discriminate between normal and alkylated globin chains. Methods: digestions of pure hemoglobin sample and of an alkylated one with Calpain I at 200:1 and 25:1 weight ratios, for 1, 5 and 18 hrs were carried out. Only the standard deviations (SD) of the ratios between alkylated globins with respect to the unmodified ones (αECH/α and bECH/b) were estimated because the aim of the study is to assess the ability of Calpain I to selectively digest the samples and, above all, to find the optimal digestion conditions and characterize obtained peptides. Results: by using a 200:1 weight ratio only peptides α(1-137), α(1-138) and b(1-142) were produced, pointing out that the first proteolytic sites are located at the C-terminus ends of both α- and b-chains. The identification of peptides obtained. C. ©. The potential health risk associated with the absorption of genotoxic agents within the organism, led to an increasing interest in the evaluation of human exposure to these agents during the last decades. Most chemical carcinogens require metabolic activation to reactive electrophiles that can modify cellular macromolecules, such as DNA and proteins. As a consequence of the exposure to a lot of chemical carcinogens, a damage of the structural integrity of DNA was observed, with the formation of a covalent carcinogen binding (carcinogen-DNA adduct) (1-4). In some cases, DNA damage is considered to be causative and directly related to tumour formation (1, 5-8); while modifications of proteins can be used to provide information about exposure, if the adduct is stable and the protein has a known life-time (9-14). In fact, protein adducts are considered a valid surrogate for DNA adducts formation, since many chemical carcinogens bind to both DNA and protein in blood with similar dose-response kinetics (3, 9, 11, 15, 16). Hemoglobin (Hb) adducts, in particular, have been found to be suitable biomarkers for evaluating exposure to toxicants (16, 17-26), due to the fact that hemoglobin is easy to sample, forms stable adducts, which are not subject to repair processes, and has a long life time (approx. 120 days in humans). Peptide mapping and tandem mass spectrometry, coupled with classical biochemical strategies, as enzymatic digestions and the modified Edman degradation, have been extensively applied to investigate protein adducts and to evaluate human exposure to toxicants (18, 27-29). Another method, based on the quantification of modified hemoglobin peptides, has been proposed, and in some cases successfully applied, for the biological monitoring of workers occupationally exposed to different classes of toxicants such as. io ni. Correspondence author: Nadia Miraglia Department of Experimental Medicine – Section of Hygiene, Occupational and Forensic Medicine – Occupational Medicine Area, Second University of Naples, Via L. De Crecchio, 7 80138, Naples, Italy E-mail: [email protected]. Prevention & Research 2014; 3(3):121-130. 121.

(2) M. Pieri et al.. na zi on. al i. bioinformatics techniques were used to identify the substrate specificities of calpains, although the accuracy of the predictions still need improvements (44). The starting point of the present work is the idea that, if Cysteine and Histidine residues of the substrate were implied in the recognition process, when alkylation occurs on these residues, Calpain would not recognize the alkylated substrate, thus permitting a selective digestion. Namely, in the case of hemoglobin, the unmodified hemoglobin would be digested, giving rise to low molecular weight peptides, while the modified one (Hb adduct) would not be digested and could be purified from the peptide mixture. Since there are few reports concerning the characterization of proteolitic sites and the reaction conditions for an optimal digestion of hemoglobin chains (and none of which involve mass spectrometry as detection technique), the first part of the work consisted in the digestion of native hemoglobin with different amounts of Calpain and different reaction times. Once the main proteolitic sites were identified, during the second part of the work a globin sample previously alkylated in vitro with epichlorohydrin (1-chloro-2,3-epoxypropane; ECH) was digested. Epichlorohydrin is widely used as raw material in the industrial synthesis of a number of glycerol and glycidiol derivatives and epoxy resins (45, 46). ECH is able to induce DNA damage (47, 48) and is considered as probably carcinogen to humans (49). The choice of ECH as alkylating agent in the development of this study was motivated by the fact that the reaction conditions for in vitro over-alkylation, as well as a punctual characterization of the alkylated amino acid residues were well-known and previously reported (22, 23, 30). The selective enrichment of the alkylated globin portion with respect to the unmodified one was evaluated through an estimation of alkylated α- and b-chains percentages in comparison with the normal ones by analysing samples before and after digestion with different amounts of Calpain I and different reaction times.. ©. C. IC. Ed. iz. io ni. In te r. epichlorohydrin, methyl bromide and butadiene (3032), putting in evidence the potentiality of this strategy. Such a procedure implies a structural characterization of the hemoglobin-carcinogen interaction, in order to identify the most reactive amino acid residue within the hemoglobin sequence. Then, a tryptic digestion of hemoglobin samples is performed and the resulting mixture is analysed by Liquid Chromatography/Electrospray-Tandem Mass Spectrometry (LC/ESI-MSMS). The major limitation of this technique is related to the low level of modified hemoglobin peptides with respect to the corresponding unmodified ones. In fact, nowadays, the levels of hemoglobin adducts found in subjects exposed to different carcinogens are in the range 0-200 pmol/g globin (18, 24), i.e. in 1 g of hemoglobin there are only 0-200 pmol of alkylated hemoglobin modified by the carcinogen. The LC/ESI-MSMS technique actually allows to achieve high instrumental sensitivity (fmol) but the alkylated peptide has to be pure, namely for the detection of hemoglobin adducts, samples must be enriched for adducts or adducts must be removed from the protein, before analysis (19). The present study was focused on the identification of an enzyme able, in principle, to discriminate between normal and alkylated globin chains, so that a selective digestion can be carried out. The basis of this choice is related to the nature of the most frequently alkylated amino acid residues within Hb. Several amino acids possess side-chains containing nucleophilic sites with reactivity towards electrophiles; this reactivity depends on the relative amount of the non-protonated form, all things considered related to the blood pH and the pKa of the group (18, 33). In particular, three amino acids in Hb show the pKa values of the nucleophilic centres in close to the blood pH: the nitrogen atoms of N-terminal Valine, the imidazole nitrogens of Histidine residues and the thiolic sulphur of Cysteins. Numerous studies have reported that at low level of concentration of the alkylating agent, Histidine and Cysteins residues are even more reactive with respect to the N-terminal Valine (14, 22, 23, 25, 30-32, 34). Therefore, in the development of a selective digestion procedure, Calpain I, a neutral calcium-dependent thiol protease (EC 3.4.22.17), seemed to be a promising choice. In particular, the μ-isozyme was preferred to the m-one, due to the minor Ca2+ demand (μM with respect to mM amounts, respectively), that is a useful feature for the subsequent LC/ESI-MS and MSMS analyses. Cysteine proteases have mechanistic similarities with serine proteases but are better nucleophiles due to the extra shell of electrons present in the sulphur of the thiol group. They require an essential Cysteine residue in the active site for hydrolysis, whose nucleophilicity is enhanced by the close proximity of an active site Histidine, which acts as a proton donor/general base. So a thiolate-imidazole charge relay diade is present, allowing a broad pH range of enzymatic activity - from 4.0 to 8.5, relating to the pKa values of Cysteine and Histidine, respectively (35). Literature studies report that Calpain was used with many proteins as substrates, with various purposes (36-41) but the structural clues of substrate recognition by Calpain are still under debate (42-44). Recently,. 122. Materials and methods Caution: Epichlorohydrin is probably carcinogenic to humans; TFA is harmful by inhalation and causes severe burns. They should be handled carefully according to appropriate environmental safety and health protocols. Epichlorohydrin was provided by Acros, Carlo Erba Reagents (Milan, Italy). Calpain I, Human Erythrocytes, was provided by Calbiochem (Merck Biosciences, Darmstadt, Germany). All other reagents were provided by Sigma-Aldrich S.r.l. The Jupiter C18 (250 X 2.0 mm, 5 μm, 300 Å) column (Phenomenex, St. Torrance, CA, USA) was used for peptide separations. HPLC gradesolvents was provided by Carlo Erba (Milan, Italy). LC/ESI-MSMS analyses were carried out using an Agilent 1100 series HPLC modular system (Palo Alto, CA, USA) and a LCQTMDECA (ThermoQuest, Finnigan, San José, CA, USA) ion trap mass spectrometer, equipped with an electrospray ion source. Prevention & Research 2014; 3(3):121-130.

(3) New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I. In vitro incubation of hemolysed red cells with ECH and globins precipitation. In te r. Hemolysed solution aliquots were suspended with 2 mL of 10mM sodium phosphate buffer (pH 7.0). ECH was added to the solution in order to have an Hb to ECH molar ratio of 1:30. The reaction mixture was left at 37 °C for 3 h, and then the solution was stored at –20 °C. The obtained globin mixture, consisting of Hb and alkylated Hb (HbECH) were precipitated with cold 5.4% HCl in acetone and washed with cold pure acetone. Then they were dried under a nitrogen stream and stored at –20 ºC. The same precipitation procedure was applied to hemolysis solution aliquots in order to obtain unmodified precipitated globins (Hb).. al i. Red cells from a healthy volunteer were washed three times with isotonic 0.9% NaCl solution and bidistillated water was added to erythrocytes to achieve a hemoglobin concentration of 5 g/100 mL. The hemolysed solution was centrifuged at 5000 rpm for 5 min in order to eliminate cellular membranes. Aliquots of 1ml were stored in freezer at –20 °C.. globin α- and b-chains sequences were searched by means of the Expasy Proteomics Server (http://us.expasy.org), the measured peptide masses were introduced, so that peptides matching the measured molecular weight values were determined. In particular, the FindPept tool was used, setting the following research parameters: average peptide masses, mass tolerance of ± 2.0 Da, Cysteins in reduced forms, without Methionines oxidized, no acidic and C-terminal residues esterified, no possible N-Acetylation or N-Formylation, without any specified proteases. When more sequences were possible, LC/ESI-MSMS analyses were carried out so that any ambiguousness was avoided in the identification of proteolytic peptides. Tandem mass spectrometry experiments were performed by using singly or doubly charged ions as first precursor ions. They were isolated in the ion trap (trapping window = 1 u) and fragmentation was induced by the application of a supplementary voltage (30%, arbitrary units) for 10 msec. Product ions mass spectra were acquired in variable mass ranges, according to the analysed peptide.. na zi on. Hemolysis. Only the standard deviations (SD) of the ratios between alkylated globins with respect to the unmodified ones (αECH/α and bECH/b) were estimated (MS Office Excell 2007) because the aim of the study is to assess the ability of Calpain I to selectively digest the samples and, above all, to find the optimal digestion conditions and characterize obtained peptides.. io ni. Enzymatic digestion of Hb and HbECH with Calpain I. Statistical analysis. Ed. iz. Aliquots of 1 mg of pure Hb or Hb alkylated as described above were digested with Calpain I at 37 °C using a solution of 0.1% TFA:(100μM CaCl2 + 100mM H3BO4) = 1:1 (v:v) as buffer, whose pH was adjusted to 7.0 by adding 30% NH4OH solution. Digestion with Calpain I was carried out by varying enzyme amounts and reaction times. In particular, globins-Calpain ratios of 200:1 and 25:1, w:w (i.e. 1 mg of globin samples were digested with 5 μg and 40 μg of Calpain, respectively) and reaction times of 1 h, 5 h and 18 h were used. Reactions were stopped by freeze drying and samples stored at –20 °C until analysis.. IC. LC/ES-MS and MSMS analysis of Calpain-digested globins. ©. C. Calpain-digested globins were fractionated by HPLC; details of chromatographic separation are reported elsewhere (31). Liquid chromatography was performed using a Phenomenex C18 (250 x 2.0 mm) column at a flow rate of 0.2 mL/min directly connected to the electrospray ion source of the mass spectrometer (heated capillary temperature = 300 °C). Data were acquired and processed using the Xcalibur program (version 1.1, ThermoQuest). Peptide mixtures were analysed in the MS mode with an acquisition range from m/z 300 to 2000. Signals of singly, doubly and triply charged ions corresponding to each peptide of interest were measured and the peptide molecular weights were worked out by the Xcalibur Biowork software (Turbosequest 1.2, ThermoQuest). The hemo-. Prevention & Research 2014; 3(3):121-130. Results Digestion of Hb: main proteolytic sites at different reaction times and Calpain amounts. Figure 1 reports the LC/ESI-MS full scan total ion chromatograms concerning the digestion of hemoglobin with Calpain at 200:1 weight ratio, for the three considered reaction times, i.e. 1, 5 and 18 hrs (panels a, b and c, respectively). In the first case, only peptides α(1-137), α(1-138) and b(1-142) were produced, thus pointing out that the first proteolytic sites are located at the C-terminus ends of both α- and b-chains. On the same enzyme amount, the increment of the reaction time resulted in an enhanced globins degree of digestion, but only when an 18-hr reaction time was used a non-negligible proteolysis was observed. In fact, even if α and b globins were the most abundant ones in the chromatogram (Fig. 1 panel c), a lot of peptides were detectable. They were identified as previously described, thus secondary proteolytic sites were identified. The complete analysis of the chromatogram allowed the identification of complementary peptides with respect to the cleavage site so that about the 100% of both α and b chains sequences was matched. The only exceptions were represented by dipeptides, which were reasonably not retained by the column. The Figure 2 reports. 123.

(4) M. Pieri et al.. na zi on. al i. Figure 1 - LC/ESI-MS total ion chromatograms of hemoglobin digested with Calpain I at 200:1 (w:w) ratio at 37 °C for 1h (panel a), 5 hrs (panel b) and 18 hrs (panel c).. iz. io ni. In te r. Figure 2 - Aminoacidic sequences of αand b-chains. Identification of primary (1) and secondary (2) proteolytic sites of hemoglobin digested by Calpain.. ©. C. IC. Ed. the primary proteolytic sites pointed out by the 18-hr reaction time experiment. It is clear that the α-chain undergoes a more marked digestion if compared to the b-chain. In general, a weight ratio between Hb and Calpain of 200:1 was not enough for obtaining a satisfactory degree of globin digestion, as stressed by the persisting of intact α- and b-globins as major compounds, even in the 18-hr experiment (Fig. 1, panel c). Digestions were repeated by increasing the amount of Calpain (25:1, weight ratio) using reaction times of 1, 5 (data not shown) and 18 hrs. As in the previous case, only when an 18 hr-reaction time was used a strong degree of proteolysis was observed (Fig. 3). The complete list of the obtained peptides is reported in Table 1. For each identified peptide, the relative retention times (RT), experimental and theoretical masses are specified. Further secondary proteolytic sites were identified and Figure 2 shows some of them, that correspond to complementary peptides with higher molecular weight; their further digestion gave rise to all the other smaller. 124. peptides reported in Table 1. In this case a higher degree of digestion for the b-chain if compared to the 200:1 (w:w) was observed; on the contrary, almost the same degree of digestion was found for the α-chain.. Digestion of Hb and HbECH mixture: selective enrichment evaluation The incubation of hemolysed solution aliquots with epichlorohydrin produces a sample containing both alkylated and unmodified hemoglobin (HbECH and Hb), whose molecular weights differ of 92 amu (or multiples, in case of higher alkylation). This sample was analysed in LC/ESI-MS (Figure 4, panel a). The chromatographic peak at RT = 66.5 min correspond to b and bECH, the one at RT = 70.0 min is constituted by α and αECH. The relative amount of alkylated α- and b-chains (αECH and bECH, respectively) with respect to the unmodified ones (α and b) was estimated. To this purpose, the ion currents of the most abundant ions of both chains were extracted from the total ion Prevention & Research 2014; 3(3):121-130.

(5) New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I. na zi on. al i. Figure 3 - LC/ESI-MS total ion chromatograms of hemoglobin digested with Calpain I at 25:1 (w:w) ratio at 37 °C for 18 hrs.. Table 1 - Identification of haemoglobin peptides from digestion with Calpain (25:1, w:w). Calculated Mass (Da). Peptide. Theoretical Mass (Da). RT (min). Calculated Mass (Da). Peptide. Theoretical Mass (Da). 6.37. 359.1 ± 0.1. b(136-139). 359.4. 39.9. 1399.9 ± 0.1 2647.3 ± 0.2 863.5 ± 0.3. b(70-82) α(43-67) α(123-130). 1400.5 2647.9 864.0. 13.52. 410.1 ± 0.1. α(40-42). 410.5. 40.3. 1286.1 ± 0.2 863.7 ± 0.2. b(13-24) α(123-130). 1286.5 864.0. 16.36. 54.2 ± 0.1. b(123-127). 540.6. 41.4. 827.5 ± 0.1 1487.1 ± 0.3. b(25-32) b(72-85). 827.9 1487.6. 17.95. 542.1 ± 0.2. α(93-97). 542.6. 42.0. 1368.9 ± 0.1. α(118-130). 1369.6. 18.55. 623.4 ± 0.2. α(88-92). 623.8. 42.5. 1158.8 ± 0.5 861.3 ± 0.3 944.0 ± 0.4. b(98-107) α(43-49) b(120-127). 1159.3 861.9 944.1. 18.9. 417.9 ± 0.1. α(64-67). 418.5. 43.0. 3469.3 ± 0.2 831.3 ± 0.1 1060.7 ± 0.3. α(43-75) b(40-45) b(38-45). 3469.8 831.9 1061.2. 22.0. 839.2 ± 0.1. 23.2. 808.6 ± 0.2. 24.56. 1073.3 ± 0.1. 25.3. 1170.9 ± 0.3. 26.6. 43.8. 1130.7 ± 0.27. α(93-102). 1131.3. 809.0. 44.6. 868.8 ± 0.2 3461.3 ± 0.4. α(103-110) b(38-69). 869.1 3461.8. b(3-12). 1074.2. 44.9. 1444.2 ± 0.2 1705.3 ± 0.4. b(3-15) b(70-85). 1444.6 1705.9. α(1-11). 1171.4. 45.3. 1680.2 ± 0.3 1165.9 ± 0.3. b(1-15) α(26-35). 1680.9 1166.4. Ed. io ni 839.9. b(62-69). 915.4 ± 0.1. b(16-24). 916.0. 45.9. 797.8 ± 0.4 1267.8 ± 0.3 1098.9 ± 0.2. α(103-109) α(76-87) α(31-39). 798.0 1268.4 1099.3. 848.4 ± 0.2 654.4 ± 0.1. b(128-135) α(82-87). 848.9 654.7. 47.0. 1651.4 ± 0.1 1274.3 ± 0.2. b(19-33) b(30-39). 1651.8 1274.5. 27.45. 632.2 ± 0.3. α(19-25). 632.6. 47.5. 1116.8 ± 0.2 1345.7 ± 0.1. b(40-48) b(38-48). 1117.3 1346.5. 27.88. 1255.4 ± 0.1. b(50-61). 1256.5. 50.4. 1307.8 ± 0.4. b(108-119). 1308.5. 28.11. 631.2 ± 0.1. b(19-24). 631.6. 50.9. 1701.7 ± 0.1 1044.6 ± 0.2 1851.8 ± 0.4. α(36-49) b(30-37) α(19-35). 1701.9 1045.3 1852.1. 28.3. 1803.0 ± 0.2 630.9 ± 0.3. α(50-67) α(76-81). 1804.0 631.7. 52.3. 1712.6 ± 0.5. b(108-122). 1713.0. 28.8. 1309.7 ± 0.2 614.2 ± 0.3. b(1-12) b(93-97). 1310.5 614.7. 54.2. 1472.5 ± 0.3 1724.8 ± 0.5. b(25-37) b(30-42). 1472.8 1725.1. 29.7. 1147.7 ± 0.2 769.4 ± 0.1. α(88-97) b(86-92). 1148.3 769.8. 54.6. 1551.6 ± 0.2. α(123-137). 1551.8. C. IC. iz. α(68-75). 26.9. ©. In te r. RT (min). continued Prevention & Research 2014; 3(3):121-130. 125.

(6) M. Pieri et al.. Table 1 - (cont.) Calculated Mass (Da). Peptide. Theoretical Mass (Da). RT (min). Calculated Mass (Da). Peptide. Theoretical Mass (Da). 30.5. 687.3 ± 0.2. α(12-18). 687.8. 65.4. 15866.8 ± 0.7 15045.7 ± 0.3 14704.6 ± 0.7. b b(1-139) b(1-135). 15867.2 15046.3 14704.9. 31.2. 2133.7 ± 0.2 2046.6 ± 0.2. b(49-69) b(50-69). 2134.5 2047.5. 66.1. 15866.5 ± 0.5. b. 15867.2. na zi on. al i. RT (min). 792.3 ± 0.1. α(131-138). 792.8. 66.6. 15866.5 ± 0.8. b. 15867.2. 1288.9 ± 0.3. α(111-122). 1289.5. 67.0. 15301.0 ± 0.5. b(1-142). 15301.6. 37.0. 1359.9 ± 0.3. α(110-122). 1360.5. 69.8. 15126.0 ± 0.5. α. 15126.4. 37.8. 2246.9 ± 0.3 853.4 ± 0.2. α(43-63) α(110-117). 2247.5 854.9. 70.5. 14591.1 ± 0.5. α(1-137). 14591.7. 38.6. 607.3 ± 0.4. α(98-102). 607.4. 71.8. 14678.2 ± 0.8. α(1-138). 14678.8. 39.6. 2646.6 ± 0.4. α(43-67). 2647.9. In te r. 33.36 36.4. Ed. iz. io ni. Figure 4 - LC/ESI-MS total ion chromatograms of an undigested mixture of Hb and HbECH (panel a). Total ion chromatograms of the same mixture digested with Calpain using a 200:1 (w:w) ratio (panel b) and 25:1 (w:w) ratio (panel c) at 37 °C for 18 hrs.. ©. C. IC. current chromatograms (TIC). Ions from [M+9H]9+ to [M+15H]15+ were considered, i.e. ions at m/z values of 1681.7, 1513.6, 1376.1, 1261.5, 1164.6, 1081.5 and 1009.4 in the case of α; ions at m/z values of 1691.9, 1522.8, 1384.5, 1269.2, 1171.6, 1088.0 and 1015.6 in the case of αECH; ions at m/z values of 1764.0, 1587.7, 1443.5, 1323.3, 1221.6, 1134.4 and 1058.8 in the case of b; ions at m/z values 1774.2, 1596.9, 1451.8, 1330.9, 1228.6, 1140.9 and 1064.9 in the case of bECH. The extraction of these ion currents from the TIC gave rise to four chromatographic peaks corresponding to α-, αECH, b-, bECH. The estimation of the relative abundance of αECH with respect to α and bECH with respect to b in the in vitro alkylated sample was obtained through the area ratio between the obtained chromatographic peaks, in particular αECH/α= 0.72 and bECH/b = 0.55. The enzymatic digestion of the same in vitro alkylated sample with Calpain I was performed at two weight ratios, 200:1 and 25:1, and at three different reaction. 126. times, 1, 5 and 18 hrs; all samples were analysed in LC/ESI-MS. As expected, the reactions at 1 and 5 hrs allowed only a low degree of digestion both for the 200:1 and 25:1 weight ratio; on the contrary a more efficient digestion was observed when reactions were conducted for 18 hrs. The LC/ESI-MS full scan chromatograms are reported in Figure 4, panels b (referred to the 200:1 weight ratio digestion of the alkylated globin samples) and c (referred to the 25:1 one). In both cases, all chromatographic peaks correspond to peptides that derived from the digestion of the unmodified hemoglobin, contained in the analysed sample. On the contrary, no peptides originated from the digestion of HbECH were found, even when the digestion was carried out for 48 hrs (data not shown). Hypothetical alkylated peptides were searched not only by analysing the total ion chromatogram but also by extracting, from the TIC, the ion currents related to their hypothetical m/z values, given by the m/z values of the peptide under inPrevention & Research 2014; 3(3):121-130.

(7) New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I. Discussion. IC. Ed. iz. al i. io ni. In te r. Proteic adducts are considered a powerful instrument for the evaluation of human exposure to carcinogenic agents and a valid surrogate of the DNA adducts. A certain number of analytical techniques are nowadays applied in the proteic adduct analysis (26, 50), as chemical and enzymatic digestions, electrophoresis or capillary liquid chromatography with different detection techniques, such as mass spectrometry and fluorescence, or immunochemical approaches, as enzymelinked immunosorbent assay (ELISA). The advancement in the instrumental performances allows high sensitivity levels, from 0.1 to 500 fmol of proteic adduct; nevertheless, there are no reports concerning the costs and time of the unavoidable purification procedure of the biological sample and the selective enrichment of the macromolecular adduct under study with respect to the unmodified proteic fraction. Over the last few years, modified Edman degradation and Raney Nickel procedures have been widely used for the biological monitoring of exposed workers (27, 51-55). Methods based on modified Edman degradation allow for the determination of adducts on the N-terminal Valine of the globin chains. However, as reported in literature, depending on the toxicant nature, Nterminal adducts could not be the most abundant ones, because of the presence of eventually more reactive sites within hemoglobin chains. In such cases, the quantification of N-terminal adducts would not reveal. low exposure levels. It has been demonstrated that, in most cases, one of the most reactive site of hemoglobin is the cysteine 93 of the b-chain (30, 56). Methods based on Raney Nickel procedure are based on the cleavage of the carbon-sulphur bond in the electrophile-cysteine adduct to form alcohols, which are extracted, suitably derivatised and analysed by gas chromatography/electron capture detection (GC/ECD) or gas chromatography/negative chemical ionizationmass spectrometry (GC/NICI-MS) (54, 57, 58). These techniques are useful in the evaluation of the exposure to aromatic toxicants, as styrene, benzene and their metabolites, but are not applied to non aromatic electrophiles. Both modified Edman degradation and Raney Nickel procedure are characterized by high sensitivity, since selective purification procedures have been developed, thus allowing the recovery and the subsequent analysis of the alkylated N-terminal residue or Cysteine-bound electrophile, that are purified from the remaining portion of globin. The determination of the internal modified peptides overcomes in principle the limitation of both modified Edman degradation and Raney Nickel procedures, since it is able to detect the carcinogenic-protein adduct level at the more reactive amino acid residue and it is generalizable to almost all electrophiles. Nevertheless, the actual applicability of the proposed procedure is subjected to the development of an efficacy purification procedure, so that the required sensitivity could be reached. With this respect, this study illustrates the preliminary results of a selective enrichment procedure focused on an enzymatic digestion with Calpain I (a cysteine protease) of hemoglobin samples containing both normal and alkylated fractions. The characterization of the main proteolytic sites in the case of pure hemoglobin as substrate revealed high reproducibility in controlled reaction condition, even if there is no specificity, since proteolysis takes place among amino acid residues of different nature. Besides, we found cleavage sites within the whole globin sequences, even if primary cleavage sites are preferentially located at the C-terminus ends. Tompa et al. investigated the recognition mechanism and sequential determinants of Calpain cleavage (42). The authors, although suggesting to investigate the influence of higher order structural elements, based their study on. na zi on. vestigation plus 92 or 46 amu ([M+H]+ and [M+2H]2+, respectively). Also in this case, no alkylated peptides were identified. The fact that HbECH was not preferentially digested by Calpain resulted in a progressive enrichment in αECH with respect to α, while the bECH/b ratio remained almost constant. In fact, for both chains the ion current of the most abundant ions within mass spectra was extracted from the total ion current chromatogram as described above. In the case of the 200:1 experiment, the αECH/α ratio was 0.79, while bECH/b was 0.50; the increasing of the enzyme amount (25:1 experiment) resulted in an increasing of αECH/α ratio, equal to 1.06, while bECH/b was equal to 0.45. Results are schematised in Table 2.. Table 2 - Relative amounts of alkylated globin chains with respect to the unmodified ones in ECH-alkylated globin samples digested with different amounts of Calpain. αECH/α±SD1. bECH/b±SD1. Undigested ECH-alkylated globins (Hb+HbECH). 0.725±0.177. 0.550±0.179. Globins:Calpain = 200:1, 1 h. 0.750±0.195. 0.543±0.210. Globins:Calpain = 200:1, 5 hrs. 0.845±0.174. 0.580±0.203. Globins:Calpain = 200:1, 18 hrs. 0.790±0.160. 0.496±0.148. Globins:Calpain = 25:1, 18 hrs. 1.056±0.158. 0.446±0.127. Globins:Calpain = 25:1, 48 hrs. 1.154±0.162. 0.475±0.133. ©. C. Sample. 1SD,. standard deviation.. Prevention & Research 2014; 3(3):121-130. 127.

(8) M. Pieri et al.. na zi on. al i. As mentioned, a part from the substrate primary structure, higher order structural elements could influence Calpain recognition and cleavage processes. That is why, even if poor selectivity was obtained between digestions of b and bECH chains, results could be different if proteolysis experiments were conducted on hemolysed samples, i.e. removing only the cellular membranes and preserving the native conformation of the protein, with the Cys 112 within the hydrophobic core and not accessible for the enzyme/ substrate recognition. The preliminary results obtained about the possibility of selectively purifying hemoglobin adducts by removing the unmodified globin fraction led us to suggest that digestion with Calpain could allow for an actual enrichment of the alkylated hemoglobin fraction present in biological samples. The subsequent purification of the proteolyses mixture by molecular sieves, with a cut off of 10000 amu, could further improve the analytical sensitivity, thus allowing the actual applicability of the internal modified peptides procedure in the evaluation of human exposure to carcinogen agents. Enzymes other than Calpain could be scrutinized for their capability to obtain a better selective degradation of modified proteins.. In te r. the primary sequence of substrates, and assigned a score to each amino acid, according to its position around the scissile bond, the so-called P2-P1 rule (59, 60). Scores are summed and the value obtained is related to the attitude for Calpain to cleave at a given position. Our results show a notable agreement with literature data as regards the prevalence of Leu and Val in the P2 position, and Lys, Arg, Tyr in P1. Moreover, the remarkable occurrence of Thr in P2 for the substrate recognition process is confirmed. On the contrary, cleavage sites found at the globins ends show low scores, as expected since they are worked out considering 11 positions around the scissile bond. The data obtained evidenced that the recognition mechanism of Calpain is not only influenced by the primary sequence of substrates but also by the reaction conditions used during the proteolysis, such as buffer, Calcium amount, temperature and time reaction. In fact, digestions carried out on precipitated globins in sodium borate at 25 °C for 10 minutes (61) brought to two cleavage sites only, Lys 11-Ala 12 (α-chain) and Lys 8-Ser 9 (b-chain): while the former was a cleavage site in our condition reaction too, the latter was not detected at all. We studied if the alkylation of Cysteine residues within the hemoglobin sequence could influence the cleavage activity of Calpain I. When digestion experiments were repeated using hemoglobin samples containing not only Hb, but both unmodified and alkylated globins (Hb and HbECH), the same proteolytic sites, hence the same peptides previously obtained, were evidenced and no alkylated peptides were found. Hemoglobin αchain contains only a Cysteine residue (Cys 104); previous studies demonstrated that such amino acid residue represents an alkylation site for ECH (22, 30). This is in line with the results obtained, which indicate that the αECH chain is not recognised as a substrate by Calpain even increasing the enzyme amount. As a consequence, a selective enrichment of αECH with respect to α was shown. Hemoglobin b-chain contains two Cysteine residues (Cys 93 and 112); under physiological condition the Cys 93 is easily alkylated, while the other one presents weak reactivity, since it lies within the hydrophobic core of the protein. The digestions with Calpain were conducted on precipitated globin samples, so, both the substrates, Hb and HbECH, did not present the typical native conformation of the protein and bchains could be recognised by the enzyme, not only by Cys 93 but through Cys 112 too. The bECH/b ratio remained nearly constant even when increasing amounts of Calpain were used, thus indicating that both b and bECH are digested. This seems to be apparently in contrast with the fact that the LC/ESI-MS full scan chromatograms did not show the presence of peptides deriving from the alkylated globin chains. The problem could actually concern the sensitivity of the MS full scan analysis itself, since the intensity of the eventually alkylated peptides could be too low for being distinguished from the chemical noise especially for small peptides, as the one formed in the Cys 93 neighbourhood. This hypothesis requires LC/ESIMSMS experiments to be confirmed.. References. Hemminki K, Dipple A, Shuker DEG, et al. 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