Journal of MASS SPECTROMETRY Accelerated communication Received: 12 February 2015 Revised: 11 March 2015 Accepted: 27 March 2015 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3599 Observation of the multiple halogenation of peptides in the electrospray ionization source Yury Kostyukevich,a,b,c Ekaterina Zhdanova,b,c Alexey Kononikhin,b,c Igor Popov,d,c Eugene Kukaevc,d and Eugene Nikolaeva,b,c,d* The chlorination of peptides and proteins is an important posttranslational modification, which is a physiological signature of an enzyme myeloperoxidase and can serve as a potential biomarker of some diseases (Parkinson’s disease, Alzheimer’s disease, etc.). The quantification of the chlorinated peptides has been very challenging in part due to their low levels and artifacts associated with sample preparation. One of the most convenient and promising methods to detect and investigate the chlorinated peptides in the biological samples is the electrospray ionization (ESI) mass spectrometry coupled to the fragmentation techniques (collision-induced dissociation and electron capture dissociation/electron transfer dissociation). We have shown that if the chlorine anions are present in the solution, then the peptide can undergo the chlorination during the ESI ionization. The effect was found to depend on the values of electric potentials of metal parts of the ESI interface. It was found that the grounding of ESI syringe results in the formation of an additional electric loop leading to the electrolytic production of Cl2 and as a consequence the hypochlorous acid inside the ESI needle. Hypochlorous acid reacts with amino groups of peptides and proteins producing chloramine or causing the protein cleavage. In the paper, it is shown on the example of the solution of the several peptides in the presence of HCl that by manipulating the ESI syringe potential, it is possible to create complexes with up to five Cl atoms for sample peptides when the ESI is operated in the positive mode. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: electrospray; peptides; chlorine; adduct formation; FT ICR Introduction J. Mass Spectrom. 2015, 50, 899–905 * Correspondence to: Eugene Nikolaev, Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninskij pr. 38 k.2, 119334 Moscow, Russia. E-mail: ennikolaev@rambler.ru a Skolkovo Institute of Science and Technology, Novaya St., 100, Skolkovo 143025, Russia b Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninskij pr. 38 k.2, 119334, Moscow, Russia c Moscow Institute of Physics and Technology, 141700, Dolgoprudnyi, Moscow Region, Russia d Emanuel Institute for Biochemical Physics, Russian Academy of Sciences, Kosygina st. 4, 119334, Moscow, Russia Copyright © 2015 John Wiley & Sons, Ltd. 899 The hypochlorous acid (HOCl) is one of the most important reactive forms of the chlorine that is formed in living organisms during the halogenating cycle of the family of mammalian heme peroxidases, mainly by myeloperoxidase.[1] HOCl has high reactivity and reacts with all major biologically important molecules: proteins, lipids, nucleic acids, carbohydrates and so on.[2–4] Many of these reactions proceed with formation of free radical intermediates. One of the most important reactions leading to free radical formation is the reaction of HOCl with amino groups. Chlorination of the NH2 group and subsequent free radical formation because of the decomposition of the chloramine leads to protein degradation, destruction of carbohydrate-containing biopolymers, inactivation of enzymes, denaturation of nucleic acids and promotion of lipid peroxidation.[5,6] The generation of free radical intermediates by HOCl and other reactive halogen species is accompanied by the development of halogenative stress, which causes a number of socially important diseases, such as cardiovascular, infectious, neurodegenerative and other diseases usually associated with inflammatory response and characterized by the appearance of biomarkers of myeloperoxidase and halogenative stress.[2,7,8] The electrospray ionization (ESI) mass spectrometry coupled to the fragmentation techniques such as collision-induced dissociation (CID) and electron capture dissociation (ECD)/electron transfer dissociation is one of the most powerful methods not only for determination of the presence of the modified proteins but also for the localization of the site of the modification.[9] Despite all advantages, there are some specific aspects of the ESI ionization that must be carefully taken care of in order to obtain adequate results. It is well-known that the interpretation of the ESI mass spectrometry results is complicated by the formation of non-covalent adduct with H2O or other polar molecules and cationization with alkali atoms (Na+, K+) or other cations such as [NH4]+. In this paper, we report that under specific conditions of the ESI, the reaction of halogenation of the peptides can occur. The ESI since its introduction in 1984[10] has become one of the most widely used ionization techniques in the biological mass spectrometry. In the ESI source, the solution of analite flows through the thin metal needle, which is kept under the high voltage. Because of the high electric field on the open side of the needle, the solution forms so-called Taylor cone, and from the tip of this cone, charge droplets emit. Those charge droplets evaporate, shrink and following several Coulomb explosions eventually produce ions. During the evaporation, charge droplets and ions interact with the surrounding atmosphere.[11–14] The flow of charged droplets is the electric current, in which generation is accompanied by the Journal of MASS SPECTROMETRY Y. Kostyukevich et al. oxidation/reduction reaction at the metal needle.[15–17] When operating ESI in positive mode in the aqueous solution, the electrochemical oxidation of water is believed to be the main charge balancing reaction[18,19] 2H2 O- > 4Hþþ 4e þ O2 (1) producing protonated molecular ions. Previously, it was reported that the corona discharge can initiate the electrochemical processes at the surface of a stainless steel electrospray capillary.[20] Such electrochemical oxidation changes the solvent composition inside the ESI needle, decreases the pH and may lead to the chemical reactions of the analite, what is strongly undesirable in bioanalytical applications.[21–23] Previously, it was shown that electrolytic oxidation can result in the emission of the metal cations from the ESI needle,[24] and that the pH changing inside ESI needle results in the conformational changes of the proteins.[25] If other anions are present in the solution, they also may participate in the charge balancing reaction. In this paper, we demonstrate that by triggering the oxidation reaction in the ESI needle, it is possible to achieve on-the-flow halogenation of the peptide. We demonstrate the formation of complexes with Cl atoms when the ESI is operated in the positive mode, the formation of Cl-peptide complexes in the negative ESI mode was described previously.[26] Methods Sample preparation In the work, we used following peptides: the ACTH Fragment 18–39 with the sequence RPVKVYPNGAEDESAEAFPLEF and molecular formula C112H165N27O36; the Αβ (1–16) peptide with the sequence (acetylated-DAEFRHDSGYEVHHQK-amidated) and molecular formula C86H122N28O28; bradykinin (1–7) with the sequence RPPGFSP and molecular formula C35H52N10O9; and the peptide P14R with and molecular formula C76H113N18O16. The solution composition was 1 : 1 mixture of water and methanol with the addition of different amounts of HCl (from 0.005% to 0.1%). Peptide concentration was equal to 100 μM. Mass spectrometer (MS) analysis All experiments were performed on an LTQ FT Ultra (Thermo Electron Corp., Bremen, Germany) mass spectrometer equipped with a 7-T superconducting magnet. Ions were generated by an IonMax Electrospray ion source (Thermo Electron Corp., Bremen, Germany) in positive ESI mode. ESI parameters were as follows: the infusion flow rate of the sample was 1 μl/min, and the ESI needle voltage was 3000 V. In order to determine the sites of chlorination, the CID and ECD fragmentation techniques were applied. Triggering the oxidation reaction was performed by changing the electric potential of the metal part of the syringe (Fig. 1). Results and discussions If the ESI solution contains a strong electrolyte, it becomes a good conductor and serves as a wire joining the needle and the metal parts of the syringe. If the syringe is grounded, the electric circuit is induced. In the presence of the HCl, the main oxidation process is as follows: 2HCl-2e - > 2Hþþ Cl2 (2) This process saturates the solution inside the ESI needle with chlorine. It is know that during the dissolving of the Cl2 in the aqueous solution, the mixture of the HCl and HOCl is formed. It was observed that when the syringe is isolated (floating potential), the total current (Itotal) becomes equal to 0.2 μA, and when the syringe is grounded, the current increases up to ~10 μA. The HOCl reacts both with a-amino group and some functional groups in the side chains of amino acids. For amino acids without a functional group in the side chain that can react with HOCl (Gly, Ala, Val and Ser), the reaction proceeds only with a-amino group with the formation of monochloramine, and dichloramine is formed in the case of excess HOCl[2] The reactions of HOCl with functional groups in the side chains of amino acids can also occur in the case of proteins and polypeptides. It means that the production of the HOCl inside the ESI needle may lead to the extensive halogenation of the peptide. Because the 900 Figure 1. The design of the ESI source with the trigger to trigger the oxidation reaction inside the ESI needle. wileyonlinelibrary.com/journal/jms Copyright © 2015 John Wiley & Sons, Ltd. J. Mass Spectrom. 2015, 50, 899–905 Journal of MASS SPECTROMETRY Halogenation of peptides in the positive ESI mode quantification of the chlorinated peptides and proteins is always complicated by their low levels and artifacts associated with sample preparation, describing a process by which these peptide adducts may form during ESI is of great interest and importance as it would identify yet another source of artifact of this analysis. Our results are presented in Fig. 2. It can be seen that when the syringe is grounded, the ACTH Fragment 18–39 forms complexes with up to five Cl atoms, Αβ (1–16) peptide forms complexes with up to three Cl atoms, Bradykinin does not seem to form complexes and the P14R peptide forms very weak complex with one Cl atom. The fact that ACTH Fragment 18–39 and Αβ (1–16) favor the complex formation and P14R and Bradykinin do not can be explained by the presence of the tyrosine (Y) residue in the ACTH Fragment 18–39 and Αβ (1–16). The tyrosine residue is known to be one of the major sites of the chlorination.[27] In order to prove the hypothesis of the chlorination of the Tyr residue, we have performed the ECD fragmentation of the (1–16) AB amyloid. In Fig. 3, it is shown that in the ECD spectrum of the peptide, the [c10 + Cl] fragment is present, the [c9 + Cl] fragment is absent, while the fragment c9 is present. It means that the chlorination occurs at the terminus residue of the c10 fragment. This is the Tyr residue. It is clear from Fig. 2 that the complexes form almost instantly after the grounding of the syringe. After the isolation of the syringe, it requires some time (~10 min) for washing away all HOCl and stabilizing the ESI. It can be seen that the most clear effect of the in-ESI chlorination is demonstrated for the ACTH Fragment 18–39, so we have performed many additional experiments to investigate the effect more accurately for this peptide. In Fig. 4(A), it can be seen that when the syringe is isolated, the main peak corresponds to the ion [M + 2H]2+. When the syringe is grounded, then complexes with three, four and five Cl are rapidly forming. After the electrical isolation of the syringe, the complexes with three, four and five Cl almost instantly disappear, but complexes with one and two Cl remain for several minutes [Fig.2(B)]. This may be explained by ‘dead volume’ of the ESI needle, in which some HOCl remains. That volume must be washed away. The increase of the flow rate results in removing these species and causes the rapid disappearing of complexes with Cl. J. Mass Spectrom. 2015, 50, 899–905 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jms 901 Figure 2. The ion map, demonstrating the formation of chlorine complexes when the syringe is grounded. (I) The syringe is isolated, and (G) the syringe is 2+ 3+ 1+ 2+ grounded. (A) [ACTH Fragment 18–39] , (B) [Αβ (1–16)] , (C) Bradykinin (756.4) and P14R (767.4) arrows show the position of the complexes with Cl and 4+ (D) [Αβ (1–16)] . Numbers indicate the number of added Cl atoms. The scale of the color represents the intensity of the corresponding peaks. Concentration of HCl is 0.1%. Journal of MASS SPECTROMETRY Y. Kostyukevich et al. Figure 3. The ECD fragments of the (1–16) AB amyloid peptide. Figure 4. (A) The complexes of ACTH Fragment 18–39 with chlorine. (B) The ion map, demonstrating the formation of chlorine complexes when the syringe is grounded. (I) The syringe is isolated and (G) the syringe is grounded. The scale of the color represents the intensity of the corresponding peaks. Concentration of HCl is 0.1%. The use of the ultrahigh resolution fourier transform ion cyclotron resonance (FT ICR) mass spectrometry demonstrated that the masses of complexes of the peptide with the Cl obey the following equation: Mcomplex þ Mpeptide þ n*MCl n*MH þ z*MH (3) 902 where MCl, MH and Mpeptide are the masses of chlorine, hydrogen and the peptide; n is the number of Cl-adducts; Mcomplex is the mass of the formed complex and z is the charge. The experimental results and the simulated spectra along with the structure of the ACTH wileyonlinelibrary.com/journal/jms Fragment 18–39 are presented in Fig. 5. This is the evidence of the formation of covalent complexes via substituting hydrogen atoms in the peptide. The concentration of the HOCl may be roughly estimated by calculating the number chlorine atoms, which are created in the solution because of the oxidation reaction. This can be calculated using Faraday’s law and taking into account that a two-electron reaction produces 1 mol of Cl2 and then 1 mol of HOCl Copyright © 2015 John Wiley & Sons, Ltd. ½ClŠ ¼ IESI =ðnFv Þ=2 (4) J. Mass Spectrom. 2015, 50, 899–905 Journal of Halogenation of peptides in the positive ESI mode MASS SPECTROMETRY Figure 5. (A) The structure of the used peptide (ACTH Fragment 18–39). (B) The measured and simulated isotopic distributions of complexes of ACTH Fragment 18–39 with chlorine. J. Mass Spectrom. 2015, 50, 899–905 Copyright © 2015 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jms 903 Figure 6. The influence of the concentration of the HCl on the formation of the chlorinated complexes of the ACTH Fragment 18–39 peptide. Journal of MASS SPECTROMETRY Y. Kostyukevich et al. Figure 7. The CID fragmentation of the complexes of ACTH Fragment 18–39 with chlorine. Here, IESI is the total current, F is the Faraday constant (9.6 × 104C/mol) and v is the volumetric flow rate out of the ESI emitter. Substituting the value of the electric current 10 μA, we obtain the rough estimation of the concentration of the HOCl present inside the ESI needle ~3 × 10 3 M. We have performed the investigation of the influence of the concentration of the HCl on the production of the chlorinated peptides. Our results are presented in Fig. 6. It can be seen that when the concentration of the HCl is 0.01%, only the traces of the chlorinated ACTH peptide can be observed. With the increase of the concentration of the HCl to 0.02%, in the spectrum appear the new peaks corresponding to the formation of complexes with one, two and three chlorine atoms. When the concentration of the HCl is 0.04%, the non-chlorinated peptide is no longer observed in the spectrum, and peaks corresponding to the formation of complexes with four and even five chlorines can be observed. In order to investigate the chlorination sites of the ACTH Fragment 18–39, we have performed the CID fragmentation of the complexes with different numbers of chlorine atoms. Our results are presented in Fig. 7. It can be seen that fragment y5 remains the Cl-free event for complex with four Cl atoms. It means that amino acid residues F, P, L and E do not form complexes with Cl under our experimental conditions. The fragment b6 has at least three possible chlorination sites, and the fragment b12 has at least four chlorination sites. The terminus residue of the b6 fragment is Tyr, which can be chlorinated. In the (6–12) part of the b12, the chlorination can occur at A or G residue. It was observed that with the increase of the number of chlorines in the complex, the efficiency of the fragment ion yield decreases. Conclusion 904 In the paper, we have demonstrated that the introduction of the additional electric loop in the ESI source by manipulating the ESI wileyonlinelibrary.com/journal/jms syringe potential allows the triggering of the electrolytic oxidation reaction inside the ESI needle. If the chlorine anions are present in the solution, then the electrochemical oxidation leads to the production of the HOCl inside the ESI needle. The HOCl reacts with the amino groups of the peptide and proteins forming the chloramines. In the paper, it is shown on the example of the solution of the several peptides in the presence of HCl that by manipulating the ESI syringe potential, it is possible to observe complexes with up to five Cl atoms for sample peptide when the ESI is operated in the positive mode. The formation of covalent complexes was proven using the ultrahigh resolution FT ICR MS and the CID fragmentation approach. Our results are important for the proteomic research because they impose limitation on the ESI parameters and demonstrate that in some cases, the modification of the peptides can occur during the mass spectrometric experiment, and those artificial modifications would interfere with the real in vivo modification that the researcher is looking for. Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Acknowledgement The work was supported by the Russian Scientific Foundation grant no. 14-24-00114. Conflict of interest Authors declare no competing financial interest. Copyright © 2015 John Wiley & Sons, Ltd. J. Mass Spectrom. 2015, 50, 899–905 Journal of MASS SPECTROMETRY Halogenation of peptides in the positive ESI mode References [1] C. C. Winterbourn. 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