Research Article Received: 14 May 2013
Revised: 25 December 2013
Accepted: 31 December 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 645–652 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6822
Electrospray ionization mass spectrometric studies on the characteristic fragmentation of Asp/cyclen conjugates Chunying Ma1, Chao Li1, Xingrong Luan1, Jin Zhang1, Renzhong Qiao1,2* and Yufen Zhao3 1
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, P. R. China State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences, Peking University Health Sciences Center, Beijing 100083, P. R. China 3 Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China 2
RATIONALE: Differentiation and structural characterization of Asp/cyclen conjugates by electrospray ionization
tandem mass spectrometry (ESI-MSn) are significantly important for their biomedical application. Hence, the present study is conducted. METHODS: The fragmentations of Asp/cyclen conjugates generated by positive ion mode electrospray ionization were examined here by low-energy collision-induced dissociation (CID). ESI-MSn spectra of cyclen were acquired to confirm cyclen contraction products derived from the studied compounds. The fragments derived from the Asp/cyclen conjugates were proved by deuterium-exchange experiments. RESULTS: Asp/cyclen conjugates displayed characteristic dissociation pathways, including cleavages of amide bonds, loss of NH3 and cyclen contraction pathways. It was observed that cleavages of C-terminal amide bonds generated b2 and b2 + H2O ions from the protonated CyclenAspAspAsp and a b1 + H2O ion from the protonated CyclenAspAsp. In addition, various cyclen contraction products were also observed. CONCLUSIONS: In ESI-MSn spectra of studied compounds, fragments of bn-1 + H2O or cyclic anhydride were generated due to facile mobilization of C-terminal or side-chain COOH protons. In addition, the cyclen contraction products were detected. These results might provide sufficient information for the identification of Asp/cyclen conjugates by mass spectrometry. Copyright © 2014 John Wiley & Sons, Ltd.
Electrospray ionization mass spectrometry (ESI-MS) offers a suitable tool and a unique physical method for structural determination in the gas-phase state. Since its introduction by Yamashita and Fenn in 1984,[1] ESI-MS has been widely used in chemical and biological research, such as detection of phosphopeptides and localization of phosphorylated sites,[2] interactions of non-covalent macromolecule-ligands,[3] and peptide sequencing.[4] Moreover, the development of ESI-MS has greatly enhanced the ability of chemists to study chemically active molecules in the gas phase and opens the door to important new applications in conventional organic chemistry analysis. In our previous work, N-phosphoryl amino acids and peptides have been extensively explored as small molecule models for the study of the intrinsic relationships between phosphoryl groups and biological molecules by ESI-MS. For example, N-terminal phosphorylation of amino acids enhanced the signal response of the amino acids up to 100-fold in ESI-MS.[5] We have also elucidated that ESI tandem mass spectrometry is very useful for the structural characterization and differentiation of a variety of amino acid and peptide derivatives.
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EXPERIMENTAL Mass spectrometry Mass spectra were acquired in positive ion mode using a Bruker ESQUIRE-LCTM ion trap spectrometer equipped with a gas nebulizer probe, capable of analyzing ions up to m/z 6000. Nitrogen (Beijing Hua Tong Jing Gas Chemical Co., Ltd) was used as drying gas at a flow rate of 4 L/min. The nebulizer gas pressure was 7.0 psi and the electrospray capillary was typically held at 4 kV. The source temperature was maintained at 300 °C. The samples, dissolved in
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* Correspondence to: R. Z. Qiao, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, P. R. China. E-mail: [email protected]
Recently, our group has reported a new family of poly (aspartic acid) grafted polyamine conjugates which could act as promising DNA cleavage agents,[6,7] giving excellent cleavage ability toward plasmid DNA.[8] In order to investigate the structure-activity relationships of this family of compounds in DNA cleavage, we designed and synthesized small molecules of aspartic acid/1,4,7,10-tetraazacyclododecane (Asp/cyclen) conjugates which have not been reported previously. Hence, the current study was designed to understand fragmentation behaviors of Asp/cyclen conjugates under electrospray ionization conditions. Meanwhile, hydrogen/deuterium (H/D)-exchange experiments were appropriately employed to rationalize the proposed fragmentation products.
C. Ma et al. methanol (Sigma-Aldrich) and methanol-d4 (Sigma-Aldrich), were continuously infused into the ESI chamber at a flow rate of 4 μL/min using a Cole-Parmer 74900 syringe pump. The scan range was from m/z 50 to 800. The MSn spectra were obtained by collision-induced dissociation (CID) of isolated precursor ions using helium buffer gas. The fragmentation amplitude values were 0.5–1.0 V and the fragmentation time was 40 ms.
Synthesis of Asp/cyclen conjugates The structures and synthetic routes of title compounds are depicted in Scheme 1 and Supplementary Schemes S1–S3 (Supporting Information), respectively. High-resolution mass spectrometry (HRMS), 1H NMR and 13C NMR spectroscopy were used to characterize the structures of Asp/cyclen conjugates.
RESULTS CID of title compounds The main fragments of protonated title compounds generated by ESI-MSn and low-energy CID are summarized in Table 1. The ESI-MSn spectra of the protonated CyclenAspAspAsp are shown in Fig. 1 and Table 1. The proposed dissociation patterns (corresponding to Fig. 1(a)) are summarized in Scheme 2. The ion at m/z 572 is formed by loss of NH3 from the precursor ion at m/z 589. The fragment ion at m/z 244 is generated by the side-chain amide bond dissociation (path B). The protonated cyclen ion at m/z 173 is derived by path C through the cleavage of the N–C bond between cyclen and -CH2- (methylene group). Interestingly, the cleavage of the C-terminal amide bond (path A) generates b2[9] and b2 + H2O ions at m/z 456 and 474, respectively (see DISCUSSION section for the proposed mechanism of this dissociation).
Scheme 1. The chemical structures CyclenAspAsp, CyclenAspAspAsp.
of
title
compounds:
CyclenAsp,
Table 1. ESI-MSn spectra data of the title compounds in MeOH Fragment ions (m/z) and relative intensity percentage (in parentheses) A Compounds CyclenAspAspAsp
CyclenAspAsp
CyclenAsp
Precursor ions, m/z 589(M + H) 572(MS2) 456(MS2) 474(MS2) 244(MS2) 359(MS3) 474(M + H) 359(MS2) 244(MS2) 173(MS2) 359(M + H) 244(MS2) 173(MS2)
bn-1 + H2O
B
572(53) 456(100)
474(18)
457(19)
359(12)
244(32) 244(100) 244(100) 244(100)
342(9) 457(23) 342(20)
359(22)
-NH3
342(8)
bn-1
C
cyclen contraction
173(6) 113(4) 173(36) 173(29) 113(26) 173(27) 173(100) 156(4),130(5),113(100),87(27),70(4) 244(100) 173(28) 113(7) 244(100) 173(25) 244(100) 173(4) 173(100) 156(3),142(4),130(4),113(39),98(11),87(12) 156(4),130(4),113(100),87(32),70(6) 244(100) 173(25) 113(6) 173(100) 156(4) 156(4),130(4),113(100),87(31),70(6)
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n = total number of residues in the protonated peptide. b2 and b2 + H2O for CyclenAspAspAsp, b1 + H2O for CyclenAspAsp.
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Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 645–652
Fragmentation studies of Asp/cyclen conjugates
Figure 1. The ESI-MSn spectra of CyclenAspAspAsp in MeOH ((a) MS/MS from the precursor ion m/z 589, (b) MS/MS/MS from the precursor ion m/z 572, (c) MS/MS/MS from the precursor ion m/z 456, (d) MS/MS/MS from the precursor ion m/z 474, (e) MS/MS/MS from the precursor ion m/z 244, (f) MS/MS/MS/MS from the precursor ion m/z 359).
Rapid Commun. Mass Spectrom. 2014, 28, 645–652
ions at m/z 342, 244 and 173 (Supplementary Fig. S2, Supporting Information) generated by the same pathways mentioned in Scheme 2. CID of 1,4,7,10-tetraazacyclododecane (cyclen) As indicated in Table 1, a novel feature of MSn spectra of the title compounds are cyclen contraction product ions observed at m/z 156, 130, 113, 87 and 70 which arise from various precursor ions, respectively. In order to further understand those cyclen contraction mechanisms, we compare the
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In order to understand the fragmentation mechanisms, ESIMSn spectra of CyclenAspAsp and CyclenAsp were acquired and studied in detail (Supplementary Figs. S1 and S2, Supporting Information). As indicated in Supplementary Fig. S1 (Supporting Information), the protonated CyclenAspAsp can generate fragment ions at m/z 244 and 173 from paths B and C, respectively, while the fragment ion at m/z 359, the protonated CyclenAsp, is derived from the path A cleavage. The ions at m/z 457 and 342 are produced by loss of NH3 from their precursor ions at m/z 474 and 359, respectively. For the protonated CyclenAsp (m/z 359), main fragments include
C. Ma et al.
Scheme 2. ESI-MS2 fragmentation pathways of the protonated CyclenAspAspAsp at m/z 589 in MeOH (corresponding to Fig. 1(a)).
Figure 2. The MSn spectra of cyclen (in MeOH).
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Scheme 3. Main fragmentations of the cyclen group.
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fragments generated from the precursor ion at m/z 173 (Supplementary Fig. S2(c), Supporting Information) with those generated from the authentic cyclen sample (Fig. 2(b)). As expected, both the spectra show the same cyclen contraction ions at m/z 156, 130, 113, 87 and 70. The main cyclen contraction pathways are elucidated in Scheme 3. The characteristic ions at m/z 156 and 113 are produced by elimination of NH3 and NH2CH2CH2NH2 from cyclen, respectively. The cyclen can also expel a molecule of CH2 = CHNH2 or piperazine to form 1,4,7-triazacyclononane (TACN) at m/z 130 or piperazine at m/z 87, respectively. The characteristic ion at m/z 70 is generated by the intramolecular elimination of piperazine and NH3 from the precursor ion at m/z 173. Compared with our previous study,[10] the new cyclen contraction ions at m/z 113 and 70 are observed in this study.
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 645–652
Fragmentation studies of Asp/cyclen conjugates CID of cyclen and title compounds through H/D-exchange experiments In MSn spectra of the studied compounds, we observed fragment ions formed by loss of NH3, amide bond cleavages or cyclen contraction pathways from various precursor ions. To clarify these observations, H/D-exchange experiments were performed by dissolving cyclen and the title compounds in MeOH-d4, respectively, and then the solutions were investigated by mass spectrometry. The amino hydrogens on cyclen were partially or fully deuterated. Hence, the mass spectra show multiple peaks (Fig. 3). As shown in Fig. 3, the cyclen gives a [M + D]+ ion at m/z 178 when the active NH is fully converted into ND. In addition, the ions at m/z 71 and 114, compared with the
non-deuterated ions at m/z 70 and 113 (Fig. 2), respectively, illustrate that there is one exchangeable hydrogen atom for the cyclen contraction product ions proposed in Scheme 3. The protonated cyclen contraction product ion at m/z 156 contains two exchangeable hydrogens (Scheme 3), which shows a fully deuterated ion at m/z 158 (Fig. 3). Protonated 1,4,7-triazacyclononane (TACN) containing four exchangeable hydrogens (Scheme 3) displays peaks at m/z 130 in MeOH (Fig. 2) and 134 in MeOH-d4 (Fig. 3). The protonated piperazine proposed in Scheme 3 displays peaks at m/z 87 in MeOH (Fig. 2) and 90 in MeOH-d4 (Fig. 3). Consequently, the data depicted in Figs. 2 and 3 are consistent with the structures proposed in Scheme 3. Hence, the cyclen contraction pathways presented in MSn spectra for title compounds were confirmed.
Figure 3. The MSn spectra of cyclen (in MeOH-d4).
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Figure 4. The MSn spectra of CyclenAspAspAsp in MeOH-d4 ((a) MS/MS from the precursor ion m/z 601, (b) MS/MS/MS from the precursor ion m/z 484, (c) MS/MS/MS/MS from the precursor ion m/z 367).
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347(16)
464(100), 581(29) 464(18) 347(23) 464(17) 347(10)
CyclenAsp (in MeOH-d4)
CyclenAspAsp(in MeOH-d4)
601(M + D) 484(MS2) 367(MS3) 484(M + D) 367(MS2) 250(MS2) 178(MS2) 367(M + D) 250(MS2) 348(MS2) 178(MS2) CyclenAspAspAsp(in MeOH-d4)
n = total number of residues in the protonated peptide. b2 and b2 + D2O for CyclenAspAspAsp, b1 + D2O for CyclenAspAsp.
367(12)
250(100)
178(9) 178(100) 178(100)
114(10) 158(12),134(8),114(50),90(27) 158(24),134(16),114(100),90(27) 114(12) 158(4),134(8),114(62),71(13) 114(89) 158(4),134(6),114(100),90(38),71(25)
90(18)
178(31) 178(36) 178(43) 178(29) 178(100) 484(22) 367(16)
250(12) 250(100) 250(100) 250(100) 250(100)
C B bn-1 + D2O -ND3 Compounds
Precursor ions, m/z
Table 2. ESI-MSn spectra data of the title compounds in MeOH-d4
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There are quite a few reports about fragmentations of Aspcontaining peptides,[11–14] most of which focus on the gasphase cleavage at aspartic acid residues in the presence of arginine residues (Arg). In the present study, a strong proton affinity residue cyclen[15] is used to mimic Arg to capture an ionizing proton. When the number of Arg units is larger than or equal to the number of ionizing protons in a peptide containing Arg and Asp, the cleavage catalyzed by the acidic hydrogen of the Asp residue occurs, therefore generating a btype ion with a cyclic anhydride structure. When the number of ionizing protons is in excess of the number of Arg, the cleavage involving the ionizing protons occurs at all backbone residues. For example, oxazolone formation by nucleophilic attack on a protonated carbonyl is via a five-membered ring, see Supplementary Scheme S4 (Supporting Information).[16] As the number of ionizing protons is equal to the number of cyclen residues in protonated CyclenAspAspAsp, it can generate one b2 ion with a cyclic anhydride structure. In addition, the MS2 spectrum exhibits a b2 ion at m/z 456 (Fig. 1(a)) from the protonated CyclenAspAspAsp. The H/D-exchange experiments for CyclenAspAspAsp (Fig. 4(a)) gave consistent results. The formation mechanism for the b2 ion is elucidated in Scheme 4 (route I). First, the ionizing proton is captured by the cyclen residue; then the side-chain COOH proton can be transferred to the nitrogen of the CyclenAspAsp-Asp amide bond, forming an intermediate as a salt-bridge (SB) structure. Then nucleophilic attack by a negatively charged hydroxyl
bn-1
DISCUSSION
A
Fragment ions (m/z) and relative intensity percentage (in parentheses)
Furthermore, deuterium-labeled experiments were also applied to testify fragments derived from Asp/cyclen conjugates. Mass spectra show multiple peaks with various intensities, including the peaks of partially and fully deuterated compounds (Fig. 4 and Supplementary Fig. S3, Supporting Information). The most abundant ions for each group of multiple peaks are listed in Table 2. As indicated in Fig. 4, CyclenAspAspAsp shows a [M + D]+ ion at m/z 601 due to the twelve exchangeable hydrogens which are completely exchanged by deuterium atoms. The fragment ion at m/z 581 is formed by loss of ND3 from the precursor ion at m/z 601. The ion at m/z 464 is consistent with the fully deuterium-labeled cyclic anhydride ion proposed in Scheme 2 which contains eight exchangeable hydrogen atoms. The [M + D]+ ion at m/z 601 can produce an ion at m/z 484 (Fig. 4(a)), 10 Da higher than the unlabeled analyte at m/z 474 (Fig. 1(a)). Tandem mass spectrometry of the precursor ion at m/z 484 produces the ion at m/z 367 as the deuterium-labeled CyclenAsp. As depicted in Table 2, CyclenAsp shows the [M + D]+ ion at m/z 367. The ion at m/z 347 is generated by elimination of ND3 from the deuterium-labeled CyclenAsp. The deuterium-labeled CyclenAspAsp at m/z 484 generates a fragment ion at m/z 464 by loss of ND3 (Supplementary Fig. S3, Supporting Information). The fragment ion at m/z 367 (Supplementary Fig. S3, Supporting Information) corresponds to the fully deuterium-labeled CyclenAsp. Since all of these fragment ions are consistent with those from unlabeled analytes, it indicates that the H/D-exchange experiments provide an effective approach for structural identification.
cyclen contraction
C. Ma et al.
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Fragmentation studies of Asp/cyclen conjugates
Scheme 4. General pathway showing the formation of the ions at m/z 456 and 474 from protonated CyclenAspAspAsp.
oxygen on the carbonyl carbon could form a five-membered ring transition state.[17] Finally, the expulsion of the C-terminal aspartic acid could generate the b2 ion (m/z 456).[18] The b2 + H2O ion generated from protonated CyclenAspAspAsp as proposed via the mechanism shown in Scheme 4 (route II) is similar to the one proposed by Paizs et al.[19] for the fragmentation of peptide [M + H]+ by loss of the C-terminal amino acid. Here a SB structure is generated due to facile mobilization of the C-terminal COOH proton.[20] From the SB intermediate the more electropositive carbonyl carbon is susceptible to nucleophilic attack by the negatively charged carboxylate oxygen to form an anhydride intermediate. Then loss of CO will generate a proton-bound dimer. The dissociation of the dimer drives the formation of the b2 + H2O ion. It is demonstrated that despite the ionizing proton being captured at the cyclen residue, the protonated CyclenAspAspAsp can be cleaved through charge-directed fragmentation pathways. This is due to facile mobilization of the C-terminal or side-chain COOH protons, thereby generating SB structures. These SB intermediates can rearrange and fragment into b2 and b2 + H2O ions. Similarly, the protonated CyclenAspAsp also gives rise to a fragment ion with one fewer aspartic acid (i.e. b1 + H2O) at m/z 359 (Supplementary Fig. S1(a), Supporting Information).
CONCLUSIONS
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Acknowledgements Support of this research by the National Nature Science Foundation of China (Nos. 21232005, 21372024, 21202005 and 21172016) is gratefully acknowledged.
REFERENCES [1] M. Yamashita, J. B. Fenn. Negative ion production with the electrospray ion source. J. Phys. Chem. 1984, 88, 4671. [2] P. J. Boersema, S. Mohammed, A. J. R. Heck. Phosphopeptide fragmentation and analysis by mass spectrometry. J. Mass Spectrom. 2009, 44, 861. [3] X. L. Chen, L. B. Qu, T. Zhang, H. X. Liu, F. Yu, Y. Z. Yu, X. C. Liao, Y. F. Zhao. The nature of phosphorylated chrysin–protein interactions involved in noncovalent complex formation by electrospray ionization mass spectrometry. Anal. Chem. 2004, 76, 211. [4] C. K. Barlow, R. A. J. O’Hair. Gas-phase peptide fragmentation: how understanding the fundamentals provides a springboard to developing new chemistry and novel proteomic tools. J. Mass Spectrom. 2008, 43, 1301. [5] Y. Chen, J. C. Zhang, J. Chen, X. Y. Cao, J. Wang, Y. F. Zhao. Sensitivity improvement of amino acids by N-terminal diisopropyloxyphosphorylation in electrospray ionization mass spectrometry. Rapid Commun. Spectrom. 2004, 18, 469. [6] J. A. Cowan. Chemical nucleases. Curr. Opin. Chem. Biol. 2001, 5, 634. [7] F. Mancin, P. Scrimin, P. Tecilla, U. Tonellato. Artificial metallonucleases. Chem. Commun. 2005, 2540. [8] C. Li, F. F. Zhao, N. Y. Huang, X. Y. Liu, Y. Liu, R. Z. Qiao, Y. F. Zhao. Metal-free DNA linearized nuclease based on PASP-polyamine conjugates. Bioconjugate Chem. 2012, 23, 1982.
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In this study, Asp/cyclen conjugates displayed characteristic fragmentation patterns, which involved cleavages of amide bonds, loss of NH3 and cyclen contraction pathways. A significant result is that the cleavages of the C-terminal amide bonds lead to the generation of b2 and b2 + H2O ions for the protonated CyclenAspAspAsp and a b1 + H2O ion for the protonated CyclenAspAsp via charge-directed fragmentation pathways, due to facile mobilization of C-terminal or sidechain COOH protons. Another novel feature of the MSn spectra of studied compounds is cyclen contraction fragment ions at m/z 156, 130, 113 and 87 produced by expulsion NH3, CH2 = CHNH2, NH2CH2CH2NH2, and piperazine from cyclen,
respectively. All the product ions are further verified by deuterium-labeled experiments. The current results will provide some contribution to the recognition of dissociation patterns and prediction of fragment ions of this class of compounds.
C. Ma et al. [9] W. Yu, J. E. Vath, M. C. Huberty, S. A. Martin. Identification of the facile gas-phase cleavage of the Asp-Pro and Asp-Xxx peptide bonds in matrix-assisted laser desorption time-offlight mass spectrometry. Anal. Chem. 1993, 65, 3015. [10] C. Li, C. Jiang, R. Z. Qiao, Y. F. Zhao. ESI-MS characteristics of N-methylpyrrole polyamide/bis-cyclen conjugate. Int. J. Mass Spectrom. 2009, 279, 176. [11] M. Rožman. Aspartic acid side chain effect – experimental and theoretical insight. J. Am. Soc. Mass. Spectrom. 2007, 18, 121. [12] C. Gu, G. Tsaprailis, L. Breci, V. H. Wysocki. Selective gasphase cleavage at the peptide bond C-terminal to aspartic acid in fixed-charge derivatives of Asp-containing peptides. Anal. Chem. 2000, 72, 5804. [13] M. J. Polce, D. Ren, C. Wesdemiotis. Dissociation of the peptide bond in protonated peptides. J. Mass Spectrom. 2000, 35, 1391. [14] S. W. Lee, H. S. Kim, J. L. Beauchamp. Salt bridge chemistry applied to gas-phase peptide sequencing: selective fragmentation of sodiated gas-phase peptide ions adjacent to aspartic acid residues. J. Am. Chem. Soc. 1998, 120, 3188. [15] W. J. Kruper, P. R. Rudolf, C. A. Langhoff. Unexpected selectivity in the alkylation of polyazamacrocycles. J. Org. Chem. 1993, 58, 3869.
[16] B. Paizs, S. Suhai. Fragmentation pathways of protonated peptides. Mass. Spectrom. Rev. 2005, 24, 508. [17] J. Qin, B. T. Chait. Preferential fragmentation of protonated gas-phase peptide ions adjacent to acidic amino acid residues. J. Am. Chem. Soc. 1995, 117, 5411. [18] R. P. Grese, R. L. Cerny, M. L. Gross. Metal ion-peptide interactions in the gas phase: a tandem mass spectrometry study of alkali metal cationized peptides. J. Am. Chem. Soc. 1989, 111, 2835 [19] B. J. Bythell, I. P. Csonka, S. Suhai, D. F. Barofsky, B. Paizs. Gas-phase structure and fragmentation pathways of singly protonated peptides with N-terminal arginine. J. Phys. Chem. B 2010, 114, 15092. [20] B. J. Bythell, S. Suhai, A. Somogyi, B. Paizs. Protondriven amide bond-cleavage pathways of gas-phase peptide ions lacking mobile protons. J. Am. Chem. Soc. 2009, 131, 14057.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s website.
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Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 645–652
Revised: 25 December 2013
Accepted: 31 December 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 645–652 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6822
Electrospray ionization mass spectrometric studies on the characteristic fragmentation of Asp/cyclen conjugates Chunying Ma1, Chao Li1, Xingrong Luan1, Jin Zhang1, Renzhong Qiao1,2* and Yufen Zhao3 1
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, P. R. China State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences, Peking University Health Sciences Center, Beijing 100083, P. R. China 3 Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China 2
RATIONALE: Differentiation and structural characterization of Asp/cyclen conjugates by electrospray ionization
tandem mass spectrometry (ESI-MSn) are significantly important for their biomedical application. Hence, the present study is conducted. METHODS: The fragmentations of Asp/cyclen conjugates generated by positive ion mode electrospray ionization were examined here by low-energy collision-induced dissociation (CID). ESI-MSn spectra of cyclen were acquired to confirm cyclen contraction products derived from the studied compounds. The fragments derived from the Asp/cyclen conjugates were proved by deuterium-exchange experiments. RESULTS: Asp/cyclen conjugates displayed characteristic dissociation pathways, including cleavages of amide bonds, loss of NH3 and cyclen contraction pathways. It was observed that cleavages of C-terminal amide bonds generated b2 and b2 + H2O ions from the protonated CyclenAspAspAsp and a b1 + H2O ion from the protonated CyclenAspAsp. In addition, various cyclen contraction products were also observed. CONCLUSIONS: In ESI-MSn spectra of studied compounds, fragments of bn-1 + H2O or cyclic anhydride were generated due to facile mobilization of C-terminal or side-chain COOH protons. In addition, the cyclen contraction products were detected. These results might provide sufficient information for the identification of Asp/cyclen conjugates by mass spectrometry. Copyright © 2014 John Wiley & Sons, Ltd.
Electrospray ionization mass spectrometry (ESI-MS) offers a suitable tool and a unique physical method for structural determination in the gas-phase state. Since its introduction by Yamashita and Fenn in 1984,[1] ESI-MS has been widely used in chemical and biological research, such as detection of phosphopeptides and localization of phosphorylated sites,[2] interactions of non-covalent macromolecule-ligands,[3] and peptide sequencing.[4] Moreover, the development of ESI-MS has greatly enhanced the ability of chemists to study chemically active molecules in the gas phase and opens the door to important new applications in conventional organic chemistry analysis. In our previous work, N-phosphoryl amino acids and peptides have been extensively explored as small molecule models for the study of the intrinsic relationships between phosphoryl groups and biological molecules by ESI-MS. For example, N-terminal phosphorylation of amino acids enhanced the signal response of the amino acids up to 100-fold in ESI-MS.[5] We have also elucidated that ESI tandem mass spectrometry is very useful for the structural characterization and differentiation of a variety of amino acid and peptide derivatives.
Rapid Commun. Mass Spectrom. 2014, 28, 645–652
EXPERIMENTAL Mass spectrometry Mass spectra were acquired in positive ion mode using a Bruker ESQUIRE-LCTM ion trap spectrometer equipped with a gas nebulizer probe, capable of analyzing ions up to m/z 6000. Nitrogen (Beijing Hua Tong Jing Gas Chemical Co., Ltd) was used as drying gas at a flow rate of 4 L/min. The nebulizer gas pressure was 7.0 psi and the electrospray capillary was typically held at 4 kV. The source temperature was maintained at 300 °C. The samples, dissolved in
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* Correspondence to: R. Z. Qiao, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, P. R. China. E-mail: [email protected]
Recently, our group has reported a new family of poly (aspartic acid) grafted polyamine conjugates which could act as promising DNA cleavage agents,[6,7] giving excellent cleavage ability toward plasmid DNA.[8] In order to investigate the structure-activity relationships of this family of compounds in DNA cleavage, we designed and synthesized small molecules of aspartic acid/1,4,7,10-tetraazacyclododecane (Asp/cyclen) conjugates which have not been reported previously. Hence, the current study was designed to understand fragmentation behaviors of Asp/cyclen conjugates under electrospray ionization conditions. Meanwhile, hydrogen/deuterium (H/D)-exchange experiments were appropriately employed to rationalize the proposed fragmentation products.
C. Ma et al. methanol (Sigma-Aldrich) and methanol-d4 (Sigma-Aldrich), were continuously infused into the ESI chamber at a flow rate of 4 μL/min using a Cole-Parmer 74900 syringe pump. The scan range was from m/z 50 to 800. The MSn spectra were obtained by collision-induced dissociation (CID) of isolated precursor ions using helium buffer gas. The fragmentation amplitude values were 0.5–1.0 V and the fragmentation time was 40 ms.
Synthesis of Asp/cyclen conjugates The structures and synthetic routes of title compounds are depicted in Scheme 1 and Supplementary Schemes S1–S3 (Supporting Information), respectively. High-resolution mass spectrometry (HRMS), 1H NMR and 13C NMR spectroscopy were used to characterize the structures of Asp/cyclen conjugates.
RESULTS CID of title compounds The main fragments of protonated title compounds generated by ESI-MSn and low-energy CID are summarized in Table 1. The ESI-MSn spectra of the protonated CyclenAspAspAsp are shown in Fig. 1 and Table 1. The proposed dissociation patterns (corresponding to Fig. 1(a)) are summarized in Scheme 2. The ion at m/z 572 is formed by loss of NH3 from the precursor ion at m/z 589. The fragment ion at m/z 244 is generated by the side-chain amide bond dissociation (path B). The protonated cyclen ion at m/z 173 is derived by path C through the cleavage of the N–C bond between cyclen and -CH2- (methylene group). Interestingly, the cleavage of the C-terminal amide bond (path A) generates b2[9] and b2 + H2O ions at m/z 456 and 474, respectively (see DISCUSSION section for the proposed mechanism of this dissociation).
Scheme 1. The chemical structures CyclenAspAsp, CyclenAspAspAsp.
of
title
compounds:
CyclenAsp,
Table 1. ESI-MSn spectra data of the title compounds in MeOH Fragment ions (m/z) and relative intensity percentage (in parentheses) A Compounds CyclenAspAspAsp
CyclenAspAsp
CyclenAsp
Precursor ions, m/z 589(M + H) 572(MS2) 456(MS2) 474(MS2) 244(MS2) 359(MS3) 474(M + H) 359(MS2) 244(MS2) 173(MS2) 359(M + H) 244(MS2) 173(MS2)
bn-1 + H2O
B
572(53) 456(100)
474(18)
457(19)
359(12)
244(32) 244(100) 244(100) 244(100)
342(9) 457(23) 342(20)
359(22)
-NH3
342(8)
bn-1
C
cyclen contraction
173(6) 113(4) 173(36) 173(29) 113(26) 173(27) 173(100) 156(4),130(5),113(100),87(27),70(4) 244(100) 173(28) 113(7) 244(100) 173(25) 244(100) 173(4) 173(100) 156(3),142(4),130(4),113(39),98(11),87(12) 156(4),130(4),113(100),87(32),70(6) 244(100) 173(25) 113(6) 173(100) 156(4) 156(4),130(4),113(100),87(31),70(6)
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n = total number of residues in the protonated peptide. b2 and b2 + H2O for CyclenAspAspAsp, b1 + H2O for CyclenAspAsp.
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Fragmentation studies of Asp/cyclen conjugates
Figure 1. The ESI-MSn spectra of CyclenAspAspAsp in MeOH ((a) MS/MS from the precursor ion m/z 589, (b) MS/MS/MS from the precursor ion m/z 572, (c) MS/MS/MS from the precursor ion m/z 456, (d) MS/MS/MS from the precursor ion m/z 474, (e) MS/MS/MS from the precursor ion m/z 244, (f) MS/MS/MS/MS from the precursor ion m/z 359).
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ions at m/z 342, 244 and 173 (Supplementary Fig. S2, Supporting Information) generated by the same pathways mentioned in Scheme 2. CID of 1,4,7,10-tetraazacyclododecane (cyclen) As indicated in Table 1, a novel feature of MSn spectra of the title compounds are cyclen contraction product ions observed at m/z 156, 130, 113, 87 and 70 which arise from various precursor ions, respectively. In order to further understand those cyclen contraction mechanisms, we compare the
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In order to understand the fragmentation mechanisms, ESIMSn spectra of CyclenAspAsp and CyclenAsp were acquired and studied in detail (Supplementary Figs. S1 and S2, Supporting Information). As indicated in Supplementary Fig. S1 (Supporting Information), the protonated CyclenAspAsp can generate fragment ions at m/z 244 and 173 from paths B and C, respectively, while the fragment ion at m/z 359, the protonated CyclenAsp, is derived from the path A cleavage. The ions at m/z 457 and 342 are produced by loss of NH3 from their precursor ions at m/z 474 and 359, respectively. For the protonated CyclenAsp (m/z 359), main fragments include
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Scheme 2. ESI-MS2 fragmentation pathways of the protonated CyclenAspAspAsp at m/z 589 in MeOH (corresponding to Fig. 1(a)).
Figure 2. The MSn spectra of cyclen (in MeOH).
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Scheme 3. Main fragmentations of the cyclen group.
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fragments generated from the precursor ion at m/z 173 (Supplementary Fig. S2(c), Supporting Information) with those generated from the authentic cyclen sample (Fig. 2(b)). As expected, both the spectra show the same cyclen contraction ions at m/z 156, 130, 113, 87 and 70. The main cyclen contraction pathways are elucidated in Scheme 3. The characteristic ions at m/z 156 and 113 are produced by elimination of NH3 and NH2CH2CH2NH2 from cyclen, respectively. The cyclen can also expel a molecule of CH2 = CHNH2 or piperazine to form 1,4,7-triazacyclononane (TACN) at m/z 130 or piperazine at m/z 87, respectively. The characteristic ion at m/z 70 is generated by the intramolecular elimination of piperazine and NH3 from the precursor ion at m/z 173. Compared with our previous study,[10] the new cyclen contraction ions at m/z 113 and 70 are observed in this study.
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Rapid Commun. Mass Spectrom. 2014, 28, 645–652
Fragmentation studies of Asp/cyclen conjugates CID of cyclen and title compounds through H/D-exchange experiments In MSn spectra of the studied compounds, we observed fragment ions formed by loss of NH3, amide bond cleavages or cyclen contraction pathways from various precursor ions. To clarify these observations, H/D-exchange experiments were performed by dissolving cyclen and the title compounds in MeOH-d4, respectively, and then the solutions were investigated by mass spectrometry. The amino hydrogens on cyclen were partially or fully deuterated. Hence, the mass spectra show multiple peaks (Fig. 3). As shown in Fig. 3, the cyclen gives a [M + D]+ ion at m/z 178 when the active NH is fully converted into ND. In addition, the ions at m/z 71 and 114, compared with the
non-deuterated ions at m/z 70 and 113 (Fig. 2), respectively, illustrate that there is one exchangeable hydrogen atom for the cyclen contraction product ions proposed in Scheme 3. The protonated cyclen contraction product ion at m/z 156 contains two exchangeable hydrogens (Scheme 3), which shows a fully deuterated ion at m/z 158 (Fig. 3). Protonated 1,4,7-triazacyclononane (TACN) containing four exchangeable hydrogens (Scheme 3) displays peaks at m/z 130 in MeOH (Fig. 2) and 134 in MeOH-d4 (Fig. 3). The protonated piperazine proposed in Scheme 3 displays peaks at m/z 87 in MeOH (Fig. 2) and 90 in MeOH-d4 (Fig. 3). Consequently, the data depicted in Figs. 2 and 3 are consistent with the structures proposed in Scheme 3. Hence, the cyclen contraction pathways presented in MSn spectra for title compounds were confirmed.
Figure 3. The MSn spectra of cyclen (in MeOH-d4).
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Figure 4. The MSn spectra of CyclenAspAspAsp in MeOH-d4 ((a) MS/MS from the precursor ion m/z 601, (b) MS/MS/MS from the precursor ion m/z 484, (c) MS/MS/MS/MS from the precursor ion m/z 367).
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347(16)
464(100), 581(29) 464(18) 347(23) 464(17) 347(10)
CyclenAsp (in MeOH-d4)
CyclenAspAsp(in MeOH-d4)
601(M + D) 484(MS2) 367(MS3) 484(M + D) 367(MS2) 250(MS2) 178(MS2) 367(M + D) 250(MS2) 348(MS2) 178(MS2) CyclenAspAspAsp(in MeOH-d4)
n = total number of residues in the protonated peptide. b2 and b2 + D2O for CyclenAspAspAsp, b1 + D2O for CyclenAspAsp.
367(12)
250(100)
178(9) 178(100) 178(100)
114(10) 158(12),134(8),114(50),90(27) 158(24),134(16),114(100),90(27) 114(12) 158(4),134(8),114(62),71(13) 114(89) 158(4),134(6),114(100),90(38),71(25)
90(18)
178(31) 178(36) 178(43) 178(29) 178(100) 484(22) 367(16)
250(12) 250(100) 250(100) 250(100) 250(100)
C B bn-1 + D2O -ND3 Compounds
Precursor ions, m/z
Table 2. ESI-MSn spectra data of the title compounds in MeOH-d4
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There are quite a few reports about fragmentations of Aspcontaining peptides,[11–14] most of which focus on the gasphase cleavage at aspartic acid residues in the presence of arginine residues (Arg). In the present study, a strong proton affinity residue cyclen[15] is used to mimic Arg to capture an ionizing proton. When the number of Arg units is larger than or equal to the number of ionizing protons in a peptide containing Arg and Asp, the cleavage catalyzed by the acidic hydrogen of the Asp residue occurs, therefore generating a btype ion with a cyclic anhydride structure. When the number of ionizing protons is in excess of the number of Arg, the cleavage involving the ionizing protons occurs at all backbone residues. For example, oxazolone formation by nucleophilic attack on a protonated carbonyl is via a five-membered ring, see Supplementary Scheme S4 (Supporting Information).[16] As the number of ionizing protons is equal to the number of cyclen residues in protonated CyclenAspAspAsp, it can generate one b2 ion with a cyclic anhydride structure. In addition, the MS2 spectrum exhibits a b2 ion at m/z 456 (Fig. 1(a)) from the protonated CyclenAspAspAsp. The H/D-exchange experiments for CyclenAspAspAsp (Fig. 4(a)) gave consistent results. The formation mechanism for the b2 ion is elucidated in Scheme 4 (route I). First, the ionizing proton is captured by the cyclen residue; then the side-chain COOH proton can be transferred to the nitrogen of the CyclenAspAsp-Asp amide bond, forming an intermediate as a salt-bridge (SB) structure. Then nucleophilic attack by a negatively charged hydroxyl
bn-1
DISCUSSION
A
Fragment ions (m/z) and relative intensity percentage (in parentheses)
Furthermore, deuterium-labeled experiments were also applied to testify fragments derived from Asp/cyclen conjugates. Mass spectra show multiple peaks with various intensities, including the peaks of partially and fully deuterated compounds (Fig. 4 and Supplementary Fig. S3, Supporting Information). The most abundant ions for each group of multiple peaks are listed in Table 2. As indicated in Fig. 4, CyclenAspAspAsp shows a [M + D]+ ion at m/z 601 due to the twelve exchangeable hydrogens which are completely exchanged by deuterium atoms. The fragment ion at m/z 581 is formed by loss of ND3 from the precursor ion at m/z 601. The ion at m/z 464 is consistent with the fully deuterium-labeled cyclic anhydride ion proposed in Scheme 2 which contains eight exchangeable hydrogen atoms. The [M + D]+ ion at m/z 601 can produce an ion at m/z 484 (Fig. 4(a)), 10 Da higher than the unlabeled analyte at m/z 474 (Fig. 1(a)). Tandem mass spectrometry of the precursor ion at m/z 484 produces the ion at m/z 367 as the deuterium-labeled CyclenAsp. As depicted in Table 2, CyclenAsp shows the [M + D]+ ion at m/z 367. The ion at m/z 347 is generated by elimination of ND3 from the deuterium-labeled CyclenAsp. The deuterium-labeled CyclenAspAsp at m/z 484 generates a fragment ion at m/z 464 by loss of ND3 (Supplementary Fig. S3, Supporting Information). The fragment ion at m/z 367 (Supplementary Fig. S3, Supporting Information) corresponds to the fully deuterium-labeled CyclenAsp. Since all of these fragment ions are consistent with those from unlabeled analytes, it indicates that the H/D-exchange experiments provide an effective approach for structural identification.
cyclen contraction
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Fragmentation studies of Asp/cyclen conjugates
Scheme 4. General pathway showing the formation of the ions at m/z 456 and 474 from protonated CyclenAspAspAsp.
oxygen on the carbonyl carbon could form a five-membered ring transition state.[17] Finally, the expulsion of the C-terminal aspartic acid could generate the b2 ion (m/z 456).[18] The b2 + H2O ion generated from protonated CyclenAspAspAsp as proposed via the mechanism shown in Scheme 4 (route II) is similar to the one proposed by Paizs et al.[19] for the fragmentation of peptide [M + H]+ by loss of the C-terminal amino acid. Here a SB structure is generated due to facile mobilization of the C-terminal COOH proton.[20] From the SB intermediate the more electropositive carbonyl carbon is susceptible to nucleophilic attack by the negatively charged carboxylate oxygen to form an anhydride intermediate. Then loss of CO will generate a proton-bound dimer. The dissociation of the dimer drives the formation of the b2 + H2O ion. It is demonstrated that despite the ionizing proton being captured at the cyclen residue, the protonated CyclenAspAspAsp can be cleaved through charge-directed fragmentation pathways. This is due to facile mobilization of the C-terminal or side-chain COOH protons, thereby generating SB structures. These SB intermediates can rearrange and fragment into b2 and b2 + H2O ions. Similarly, the protonated CyclenAspAsp also gives rise to a fragment ion with one fewer aspartic acid (i.e. b1 + H2O) at m/z 359 (Supplementary Fig. S1(a), Supporting Information).
CONCLUSIONS
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Acknowledgements Support of this research by the National Nature Science Foundation of China (Nos. 21232005, 21372024, 21202005 and 21172016) is gratefully acknowledged.
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In this study, Asp/cyclen conjugates displayed characteristic fragmentation patterns, which involved cleavages of amide bonds, loss of NH3 and cyclen contraction pathways. A significant result is that the cleavages of the C-terminal amide bonds lead to the generation of b2 and b2 + H2O ions for the protonated CyclenAspAspAsp and a b1 + H2O ion for the protonated CyclenAspAsp via charge-directed fragmentation pathways, due to facile mobilization of C-terminal or sidechain COOH protons. Another novel feature of the MSn spectra of studied compounds is cyclen contraction fragment ions at m/z 156, 130, 113 and 87 produced by expulsion NH3, CH2 = CHNH2, NH2CH2CH2NH2, and piperazine from cyclen,
respectively. All the product ions are further verified by deuterium-labeled experiments. The current results will provide some contribution to the recognition of dissociation patterns and prediction of fragment ions of this class of compounds.
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[16] B. Paizs, S. Suhai. Fragmentation pathways of protonated peptides. Mass. Spectrom. Rev. 2005, 24, 508. [17] J. Qin, B. T. Chait. Preferential fragmentation of protonated gas-phase peptide ions adjacent to acidic amino acid residues. J. Am. Chem. Soc. 1995, 117, 5411. [18] R. P. Grese, R. L. Cerny, M. L. Gross. Metal ion-peptide interactions in the gas phase: a tandem mass spectrometry study of alkali metal cationized peptides. J. Am. Chem. Soc. 1989, 111, 2835 [19] B. J. Bythell, I. P. Csonka, S. Suhai, D. F. Barofsky, B. Paizs. Gas-phase structure and fragmentation pathways of singly protonated peptides with N-terminal arginine. J. Phys. Chem. B 2010, 114, 15092. [20] B. J. Bythell, S. Suhai, A. Somogyi, B. Paizs. Protondriven amide bond-cleavage pathways of gas-phase peptide ions lacking mobile protons. J. Am. Chem. Soc. 2009, 131, 14057.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s website.
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