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Toyonaka, Osaka 560-0043, Japan, and Life Science Laboratory, Shimadzu Corporation, Kyoto 604-8511, Japan. The novel N-terminal labeling method using ...
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Anal. Chem. 2005, 77, 6618-6624

Distinction of Leu and Ile Using a Ruthenium(II) Complex by MALDI-LIFT-TOF/TOF-MS Analysis Akihiro Ito,† Taka-aki Okamura,*,† Hitoshi Yamamoto,† Norikazu Ueyama,*,† Kojiro Ake,‡ Ryoji Masui,‡ Seiki Kuramitsu,‡ and Susumu Tsunasawa§

Department of Macromolecular Science and Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan, and Life Science Laboratory, Shimadzu Corporation, Kyoto 604-8511, Japan

The novel N-terminal labeling method using a ruthenium(II) complex derivative characteristically indicated an and dn (N-terminal) fragment ions in high sensitivity by MS/ MS analysis (MALDI-LIFT or ESI-CID). Although these fragment ions depended on a fragmentation process by MS/MS analytical methods to some degree, each case indicated similar side-chain cleavage patterns. The labeling method allows accurate distinction of amino acid residues by MS/MS analysis even if the residues are structural isomers such as leucine and isoleucine. The method was applied to long-chain peptides and provided easy and rapid N-terminal sequencing. The current strategy for protein amino acid sequencing involves enzymatic digestion of proteins followed by mass spectrometry (MS) of the resulting peptide fragmentssfor example, matrix-assisted laser desorption/ionization (MALDI) postsource decay MS,1-8 electrospray ionization (ESI) MS/MS,9-12 and Fourier transform ion cyclotron resonance MS.13-17 Peptide mass fingerprinting and peptide sequence tagging have been widely used to identify the proteins, whose sequences are available in * To whom correspondence should be addressed. E-mail: tokamura@ chem.sci.osaka-u.ac.jp (T.O.); [email protected] (N.U.). Fax: +81 -6-6850-5474. † Department of Macromolecular Science, Osaka University. ‡ Department of Biology, Osaka University. § Shimadzu Corp. (1) Medzihradszky, K. F.; Campbell, J. M.; Baldwin, M. A.; Falick, A. M.; Juhasz, P.; Vestal, M. L.; Burlingame, A. L. Anal. Chem. 2000, 72, 552-558. (2) Yergey, A. L.; Coorssen, J. R.; Backlund, P. S., Jr.; Blank, P. S.; Humphrey, G. A.; Zimmerberg, J.; Campbell, J. M.; Vestal, M. L. J. Am. Soc. Mass. Spectrom. 2002, 13, 784-791. (3) Qian, X.; Zhou, W.; Khaledi, M. G.; Tomer, K. B. Anal. Biochem. 1999, 274, 174-180. (4) Yates, J. R. J. Mass Spectrom. 1998, 33, 1-19. (5) Marvina, L. F.; Robertsb, M. A.; Faya, L. B. Clin. Chim. Acta 2003, 337, 11-21. (6) Suckau, D.; Resemann, A.; Schuerenberg, M.; Hufnagel, P.; Franzen, J.; Holle, A. Anal. Bioanal. Chem. 2003, 376, 952-965. (7) Spengler, B. J. Mass Spectrom. 1997, 32, 1019-1036. (8) Warscheid, B.; Fenselau, C. Anal. Chem. 2003, 75, 5618-5627. (9) Griffiths, W. J.; Jonsson, A. P.; Liu, S.; Rai, D. K.; Wang, Y. Biochem. J. 2001, 355, 545-561. (10) Morera, V.; Gomez, J.; Besada, V.; Estrada, R.; Pons, T.; Alvarez, C.; Tejuca, M.; Padron, G.; Lanio, M. E.; Pazos, F. Biotecnologia Aplicada 1995, 12, 169-170. (11) Zhu, H.; Bilgin, M.; Snyder, M. Annu. Rev. Biochem. 2003, 72, 783-812. (12) Hu, S.; Zhang, L.; Newitt, R.; Aebersold, R.; Kraly, J. R.; Jones, M.; Dovichi, N. J. Anal. Chem. 2003, 75, 3502-3505. (13) Zabrouskov, V.; Giacomelli, L.; van Wijk, K. J.; McLafferty, F. W. Mol. Cell. Proteomics 2003, 1253-1260.

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databases.4,18-21 Sometimes, these methods lead to ambiguous protein identification because of the complicated mixture of enzymatic peptides from comigrating proteins, unknown proteins with no matches in the database,22-24 and difficulty of accurate distinction of structural isomers (e.g., leucine and isoleucine) or similar molecular weight amino acid residues (e.g., glutamine and lysine). To characterize precisely the protein structure and function requires unambiguous protein identification. The cleavage of the Cβ-Cγ bond (namely, dn or wn fragment ions) in MS/MS analysis lets one distinguish Leu/Ile isomers or Gln/Lys isobars.15,25,26 Only a few examples have been published in which low-energy collision-induced dissociation (CID) is capable of distinguishing leucine and isoleucine.26-33 Turecˇek and coworkers reported that the addition of metal ions to peptide solutions enhances the sensitivity of MS/MS spectra in ESI(14) O’Brien, D. P.; Kirkpartrick, P. N.; O’Brien, S. W.; Staroske, T.; Richardson, T. I.; Evans, D. A.; Hopkinson, A.; Spencer, J. B.; Williams, D. H. Chem. Commun. 2000, 103-104. (15) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528-9533. (16) Kjeldsen, F.; Haselmann, K. F.; Sørensen, E. S.; Zubarev, R. A. Anal. Chem. 2003, 75, 1267-1274. (17) Demirev, P. A.; Ramirez, J.; Fenselau, C. Anal. Chem. 2001, 73, 57255731. (18) Jensen, O. N.; Podtelejnikov, A. V.; Mann, M. Anal. Chem. 1997, 69, 47414750. (19) Gharahdaghi, F.; Weinberg, C. R.; Meagher, D. A.; Imai, B. S.; Mische, S. M. Electrophoresis 1999, 20, 601-605. (20) Katayama, H.; Nagasu, T.; Oda, Y. Rapid Commun. Mass Spectrom. 2001, 15, 1416-1421. (21) Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 4390-4399. (22) Keough, T.; Lacey, M. P.; Youngquist, R. S. Rapid Commun. Mass Spectrom. 2002, 16, 1003-1015. (23) Hellman, U.; Bhikhabhai, R. Rapid Commun. Mass Spectrom. 2002, 16, 1851-1859. (24) Chen, P.; Nie, S.; Mi, W.; Wang, X.-C.; Liang, S.-P. Rapid Commun. Mass Spectrom. 2004, 18, 191-198. (25) Zaia, J.; Biemann, K. J. Am. Soc. Mass Spectrom. 1995, 6, 428-436. (26) Bosse´e, A.; Fournier, F.; Tasseau, O.; Bellier, B.; Tabet, J.-C. Rapid Commun. Mass Spectrom. 2003, 17, 1229-1239. (27) Seymour, J. L.; Turecˇek, F. J. Mass Spectrom. 2000, 35, 566-571. (28) Vaisar, T.; Gatlin, C. L.; Rao, R. D.; Seymour, J. L.; Turecˇek, F. J. Mass Spectrom. 2001, 36, 306-316. (29) Gatlin, C. L.; Turecˇek, F. J. Mass Spectrom. 2000, 35, 172-177. (30) Wee, S.; O’Hair, R. A. J.; McFadyen, W. D. Rapid Commun. Mass Spectrom. 2002, 16, 884-890. (31) Tao, W. A.; Wu, L.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 2001, 12, 490-496. (32) Nemirovskiy, O. V.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1996, 7, 977980. (33) Nemirovskiy, O. V.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1998, 9, 12851292. 10.1021/ac050534o CCC: $30.25

© 2005 American Chemical Society Published on Web 09/17/2005

LIFT-TOF/TOF-MS6,43 analysis. In addition, ESI-CID was also employed to the distinction of Leu/Ile isomers using 〈Ru〉-COlabeling method. The characterization method was applied to a long-chain peptide to establish a N-terminal sequencing procedure.

Figure 1. Active ester of bis(terpyridine)ruthenium(II) complex (1).

CID.27-29 In these cases, a ternary bidentate amino acid complex with Cu2+ in the gas phase underwent side-chain fragmentations that distinguished Leu/Ile isomers. Specific N-terminal modifications improve the sensitivity of the desired fragment ions and simplify the MS spectrum.25,34 The active esters of nicotinic acid derivatives35,36 have been reported to show a characteristic isotope pattern, giving enhanced signals of N-terminal fragment ions (an and dn ions) in MS/MS analysis. Although these methods enhance the sensitivity of desired fragment ions, strict distinction between N- and C-terminal fragment ions and elimination of undesired peaks are very difficult to achieve. Recently, we demonstrated protein sequencing using an active ester of bis(terpyridine)ruthenium(II) derivative (Figure 1) as a novel N-terminal labeling reagent.37,38 The reagent has a characteristic isotope pattern and yields highly intense peaks in MS analysis.37-41 By using this reagent for amino acid sequencing of peptides or proteins, we selectively detected N-terminal fragment ions with no C-terminal fragment ion. N-Terminal labeling with the reagent 〈Ru〉-CO achieves rapid determination of the Nterminal amino acid sequence.38 The detection limit is at the ∼10fmol level, which is enough to analyze each visible spot in twodimensional electrophoresis separation, whereas detection gel staining requires a detection threshold of ∼100 fmol.42 In this paper, the side-chain cleavage of 〈Ru〉-CO-labeled amino acid residue was detected using their methyl esters and dipeptides as simple models of N-terminal amino acid sequences by MALDI(34) Roth, K. D. W.; Huang, Z.-H.; Sadagopan, N.; Watson, J. T. Mass Spectrom. Rev. 1998, 17, 255-274. (35) Miyagi, M.; Nakao, T.; Nakazawa, T.; Kato, I.; Tsunasawa, S. Rapid Commun. Mass Spectrom. 1998, 12, 603-608. (36) Mu ¨ nchbach, M.; Quadroni, M.; Miotto, G.; James, P. Anal. Chem. 2000, 72, 4047-4057. (37) Ueyama, N.; Okamura, T.; Norioka, S.; Nakazawa, T.; Kuyama, H.; Ando, E. U.S. patent 10,614,324, 2004. (38) Okamura, T.; Iwamura, T.; Ito, A.; Kaneko, M.; Yamaguchi, M.; Yamamoto, H.; Ueyama, N.; Kuyama, H.; Ando, E.; Norioka, S.; Nakazawa, T.; Masui, R.; Kuramitsu, S. Chem. Lett. 2005, 34, 332-333. (39) Okamura, T.; Ueyama, N.; Iwamura, T.; Ikemori, M.; Kaneko, M.; Masui, K.; Yamamoto, H.; Yamaguchi, M.; Kuyama, H.; Ando, E.; Tsunasawa, S. J. Inorg. Biochem. 2003, 96, 204. (40) Okamura, T.; Iwamura, Y.; Seno, S.; Yamamoto, H.; Ueyama, N. J. Am. Chem. Soc. 2004, 15972-15973. (41) Kaneko, M.; Masui, R.; Ake, K.; Kousumi, Y.; Kuramitsu, S.; Yamaguchi, M.; Kuyama, H.; Ando, E.; Norioka, S.; Nakazawa, T.; Okamura, T.; Yamamoto, H.; Ueyama, N. J. Proteome Res. 2004, 3, 983-987. (42) Katayama, H.; Satoh, K.; Takeuchi, M.; Deguchi-tawarada, M.; Oda, Y.; Nagasu, T. Rapid Commun. Mass Spectrom. 2003, 17, 1071-1078.

EXPERIMENTAL SECTION Materials. [(tpy)Ru(tpyC6H4COONSu)](PF6)2 was synthesized by the same procedure reported in the previous paper.38 Watersoluble carbodiimide (WSCD), N-hydroxysuccinimide (HONSu), HCl‚H-Leu-OMe, HCl‚H-Ile-OMe, HCl‚H-Leu-Gly-OEt, HCl‚H-IleGly-OEt, HCl‚H-Val-OMe, HCl‚H-Ala-OMe, and H-Ile-Ser-bradykinin-OH were used in commercial grade. N,N-Dimethylformamide (DMF) and acetonitrile were purified by distillation. Synthesis. [(tpy)Ru(tpyC6H4CO-Leu-OMe)](PF6)2. [(tpy)Ru(tpyC6H4COONSu)](PF6)2 (20 mg, 0.019 mmol) and HCl‚HLeu-OMe (20 mg, 0.11 mmol) were dissolved in DMF (0.1 mL) under an Ar atmosphere at 0 °C. Triethylamine (0.2 mL) was added to the solution at 0 °C. After stirring overnight at room temperature, the solution was concentrated to dryness under reduced pressure. The residue was washed with ethyl acetate several times and dissolved in acetonitrile. The addition of ethyl acetate to the solution yielded a reddish-orange precipitate, which was collected and dried over P2O5 under reduced pressure. The amount yielded was 12 mg (55%). 1H NMR (303 K, dimethyl sulfoxide-d6): δ 0.97 (m, 6H, δ-CH3), 1.63 (t, 2H, J ) 7.2 Hz, β-CH2), 1.76 (m, 1H, γ-CH), 3.70 (s, 3H, OMe), 4.47 (br, 1H, R-CH), 6.03 (br, 1H, NH), 7.26 (m, 4H, H5A, H5B), 7.43 (d, 2H, J ) 4.8 Hz, H6B), 7.54 (d, 2H, J ) 5.6 Hz, H6A), 8.05 (m, 6H, Hm, H4A, H4B), 8.26 (d, 2H, J ) 8.4 Hz, Ho), 8.55 (m, 1H, H4′A), 8.83 (d, 2H, J ) 8.0 Hz, H3B), 9.11 (d, 4H, J ) 8.0 Hz, H3A, H3′B), 9.52 (s, 2H, H3′A). ESI-MS: [M]2+ m/z 407.7 (calcd 407.7). [(tpy)Ru(tpyC6H4CO-Ile-OMe)](PF6)2. This compound was synthesized by a method similar to that described for [(tpy)Ru(tpyC6H4CO-Leu-OMe)](PF6)2. Yield 16 mg (73%). 1H NMR (303 K, dimethyl sulfoxide-d6): δ 1.21 (m, 6H, γ-CH3, δ-CH3), 2.35 (m, 2H, γ-CH2), 3.71 (s, 3H, OMe), 4.03 (m, 1H, β-CH), 4.47 (br, 1H, R-CH), 6.03 (br, 1H, NH), 7.26 (m, 4H, H5A, H5B), 7.43 (d, 2H, J ) 4.8 Hz, H6B), 7.53 (d, 2H, J ) 5.6 Hz, H6A), 8.05 (m, 6H, Hm, H4A, H4B), 8.26 (d, 2H, J ) 8.4 Hz, Ho), 8.54 (m, 1H, H4′A), 8.83 (d, 2H, J ) 8.0 Hz, H3B), 9.11 (d, 4H, J ) 8.0 Hz, H3A, H3′B), 9.52 (s, 2H, H3′A). ESI-MS: [M]2+ m/z 407.7 (calcd 407.7). [(tpy)Ru(tpyC6H4CO-Leu-Gly-OEt)](PF6)2. This compound was synthesized by a method similar to that described for [(tpy)Ru(tpyC6H4CO-Leu-OMe)](PF6)2. Yield 7 mg (86%). 1H NMR (303 K, dimethyl sulfoxide-d6): δ 0.97 (m, 6H, Leu-δ-CH3), 1.00 (t, 3H, OCH2CH3), 1.63 (t, 2H, J ) 7.2 Hz, Leu-β-CH2), 1.76 (m, 1H, γ-CH), 3.70 (m, 2H, OCH2CH3), 4.16 (d, 2H, Gly-CH2), 4.47 (br, 1H, LeuR-CH), 6.03 (br, 1H, Gly-NH), 6.43 (br, 1H, Leu-NH), 7.26 (m, 4H, H5A, H5B), 7.43 (d, 2H, J ) 4.8 Hz, H6B), 7.54 (d, 2H, J ) 5.6 Hz, H6A), 8.05 (m, 6H, Hm, H4A, H4B), 8.26 (d, 2H, J ) 8.4 Hz, Ho), 8.55 (m, 1H, H4′A), 8.83 (d, 2H, J ) 8.0 Hz, H3B), 9.11 (d, 4H, J ) 8.0 Hz, H3A, H3′B), 9.52 (s, 2H, H3′A). ESI-MS: [M]2+ m/z 443.2 (calcd 443.1). [(tpy)Ru(tpyC6H4CO-Ile-Gly-OEt)](PF6)2. This compound was synthesized by a method similar to that described for (43) Schnaible, V.; Wefing, S.; Resemann, A.; Sukau, D.; Bu ¨ ker, A.; WolfKu ¨ mmeth, S.; Hoffmann, D. Anal. Chem. 2002, 74, 4980-4988.

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Figure 2. Predicted fragment ions caused by the cleavage of the side chain.

Scheme 1. Synthetic Route of Amino Acid-Ru(II) Complex

[(tpy)Ru(tpyC6H4CO-Leu-OMe)](PF6)2. Yield 7 mg (86%). 1H NMR (303 K, dimethyl sulfoxide-d6): δ 1.00 (t, 3H, OCH2CH3), 1.21 (m, 6H, Ile-γ-CH3, Ile-δ-CH3), 2.35 (m, 2H, Ile-γ-CH2), 3.71 (m, 2H, OCH2CH3), 4.03 (m, 1H, Ile-β-CH), 4.16 (d, 2H, Gly-CH2), 4.47 (br, 1H, Ile-R-CH), 6.03 (br, 1H, Gly-NH), 6.45 (br, 1H, Ile-NH), 7.25 (m, 4H, H5A, H5B), 7.44 (d, 2H, J ) 4.8 Hz, H6B), 7.52 (d, 2H, J ) 5.6 Hz, H6A), 8.02 (m, 6H, Hm, H4A, H4B), 8.30 (d, 2H, J ) 8.4 Hz, Ho), 8.55 (m, 1H, H4′A), 8.84 (d, 2H, J ) 8.0 Hz, H3B), 9.10 (d, 4H, J ) 8.0 Hz, H3A, H3′B), 9.52 (s, 2H, H3′A). ESI-MS: [M]2+ m/z 443.1 (calcd 443.1). [(tpy)Ru(tpyC6H4CO-Val-OMe)](PF6)2. This compound was synthesized by a method similar to that described for [(tpy)Ru(tpyC6H4CO-Leu-OMe)](PF6)2. Yield 16 mg (73%). 1H NMR (303 K, dimethyl sulfoxide-d6): δ 1.25 (m, 6H, γ-CH3), 2.35 (m, 1H, β-CH), 3.45 (s, 3H, OMe), 4.03 (m, 1H, β-CH), 4.47 (br, 1H, R-CH), 6.09 (br, 1H, NH), 7.26 (m, 4H, H5A, H5B), 7.42 (d, 2H, J ) 4.8 Hz, H6B), 7.53 (d, 2H, J ) 5.6 Hz, H6A), 8.05 (m, 6H, Hm, H4A, H4B), 8.33 (d, 2H, J ) 8.4 Hz, Ho), 8.53 (m, 1H, H4′A), 8.82 (d, 2H, J ) 8.0 Hz, H3B), 9.09 (d, 4H, J ) 8.0 Hz, H3A, H3′B), 9.49 (s, 2H, H3′A). ESI-MS: [M]2+ m/z 400.6 (calcd 400.6). [(tpy)Ru(tpyC6H4CO-Ala-OMe)](PF6)2. This compound was synthesized by a method similar to that described for [(tpy)Ru(tpyC6H4CO-Leu-OMe)](PF6)2. Yield 16 mg (73%). 1H NMR (303 K, dimethyl sulfoxide-d6): δ 1.50 (d, 3H, J ) 4.2 Hz, β-CH3), 3.58 (s, 3H, OMe), 4.56 (br, 1H, R-CH), 6.09 (br, 1H, NH), 7.26 (m, 4H, H5A, H5B), 7.42 (d, 2H, J ) 4.8 Hz, H6B), 7.53 (d, 2H, J ) 5.6 Hz, H6A), 8.05 (m, 6H, Hm, H4A, H4B), 8.33 (d, 2H, J ) 8.4 Hz, Ho), 8.53 (m, 1H, H4′A), 8.82 (d, 2H, J ) 8.0 Hz, H3B), 9.09 (d, 4H, J ) 8.0 Hz, H3A, H3′B), 9.49 (s, 2H, H3′A), 9.83 (br, 1H, NH2). ESI-MS: [M]2+ m/z 386.6 (calcd 386.6). [(tpy)Ru(tpyC6H4CO-Ile-Ser-bradykinin)](PF6)2. This compound was synthesized by a method similar to that described for [(tpy)Ru(tpyC6H4CO-Leu-OMe)](PF6)2. Yield 0.25 mg (65%). ESIMS: [M]3+ m/z 643.7 (calcd 643.9), [M]4+ m/z 483.1 (calcd 483.2), [M]5+ m/z 387.0 (calcd 386.8). Physical Measurements. MALDI spots were made by condensation of a mixture of sample solution (5 µL, 10 pmol µL-1) with 5 µL of matrix solution. One microliter of the mixed solution was deposited on a stainless steel plate and dried under ambient conditions. R-Cyano-4-hydroxycinnamic acid was saturated in 50% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid, which was used as a matrix solution. MALDI-LIFT spectra were recorded in positive mode on a Bruker Ultraflex TOF/TOF mass spectrometer controlled by the 6620 Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

Figure 3. MALDI-LIFT spectra of (a) 〈Ru〉-CO-Val-OMe and (b) 〈Ru〉-CO-Ala-OMe.

Flexcontrol 2.2 software package. External calibration of MALDI mass spectra was carried out using singly charged monoisotopic peaks of a mixture of human angiotensin II (m/z 1046.54), angiotensin I (m/z 1296.68), bombesin (m/z 1619.82), and adrenocorticotropic hormone (ACTH clip 1-17) (m/z 2093.08). Metastable ions were analyzed that were generated by laser-induced decomposition of the selected precursor ions. No additional collision gas was applied. Precursor ions were accelerated to 8 kV and selected in a timed ion gate. The fragments were further accelerated to 19 kV in the LIFT cell, and their masses were analyzed after the ion reflector passage. ESI-MS spectra were recorded on a Finnigan MAT LCQ ion trap mass spectrometer using an acetonitrile solution. 1H NMR spectra were recorded on a JEOL JNM-LA500 in dimethyl sulfoxide-d6 solution at 303 K. RESULTS AND DISCUSSION Fragmentation of Aliphatic Amino Acids. A series of 〈Ru〉CO-labeled aliphatic amino acid residues including structural

Figure 4. MALDI-TOF/TOF-MS spectra of 〈Ru〉-CO-Leu-OMe (a) and 〈Ru〉-CO-Ile-OMe (b).

isomers of leucine and isoleucine (Scheme 1) was measured by MALDI-LIFT to analyze their fragmentation pattern (Figure 2). Determining which C-C bond in the side chain is cleaved is simple because it consists of the C-C bond only. MALDI-LIFT spectra of 〈Ru〉-CO-Ala-OMe and 〈Ru〉-CO-ValOMe are shown in Figure 3. The side-chain cleavage was formed predominantly between the Cβ-Cγ bond but not the CR-Cβ bond. Valine showed d1 (m/z 727.02, calcd 726.79) and d1 - CH3 (m/z 712.83, calcd 713.15) fragment ions arising from the cleavage of the Cβ-Cγ bond. Alanine yielded no fragment peak by the sidechain cleavage. Each residue indicates an a1 fragment ion (Val: m/z 741.99, calcd 741.81. Ala: m/z 712.65, calcd 713.15) and showed no ion formed by the cleavage of the CR-Cβ bond (Figures 2 and 3) around m/z 700. Figure 4 shows the MALDI-TOF/TOF-MS spectra of 〈Ru〉-COLeu-OMe and 〈Ru〉-CO-Ile-OMe. Both compounds exhibit almost the same m/z value (calcd 815.22) and some in-source fragment ions. To distinguish them from each other, we analyzed the sidechain cleavage patterns of these residues by MALDI-LIFT (Figure 5). Proposed structures for the obtained fragment ions are shown in Figure 6. Table 1 lists the observed values in Figure 5 with calculated values. These fragmentations are clearly distinguishable from each other by the characteristic peak at m/z 727. Figure 5a showed no fragment peak by loss of two methyl groups around m/z 727. On the other hand, the spectrum of 〈Ru〉-CO-Ile-OMe (Figure 5b) exhibited the loss of the ethyl group (m/z 727.45, calcd 726.79). The fragment peak at m/z 727.45 is in perfect accordance with its characteristic isotope pattern with simulated spectrum (Figure 5c). Major fragment peaks are caused by the cleavage of the Cβ-Cγ bond. The cleavage of the CR-Cβ bond was not found. Using the 〈Ru〉-CO-labeling method, these masses

Figure 5. MALDI-LIFT spectra and its expanded spectra of 〈Ru〉CO-Leu-OMe (a), 〈Ru〉-CO-Ile-OMe (b), and simulated spectrum of d1 fragment of 〈Ru〉-CO-Ile-OMe (c).

of the fragment peaks shift to high mass range, which is the highresolution region in MALDI-MS or MS/MS. Thus, these fragment peaks were detected as highly sensitive and the method enabled us to analyze the fragmentation pattern in detail. The dipeptides, 〈Ru〉-CO-Leu-Gly-OEt and 〈Ru〉-CO-Ile-Gly-OEt, yielded fragmentation (Figure 7) similar to that of their methyl ester derivatives (Figure 5). 〈Ru〉-CO-Leu-Gly-OEt showed no Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

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Figure 6. Expected fragment ions obtained from the cleavage of 〈Ru〉-CO-Leu-OMe (a) and 〈Ru〉-CO-Ile-OMe (b).

Figure 8. ESI-CID spectra of 〈Ru〉-CO-Leu-OMe (a) and 〈Ru〉-COIle-OMe (b). Figure 7. MALDI-LIFT spectra and its expanded spectra of 〈Ru〉CO-Leu-Gly-OEt (a) and 〈Ru〉-CO-Ile-Gly-OEt (b).

fragment peak around m/z 727 (Figure 7a). In Figure 7b, 〈Ru〉CO-Ile-Gly-OEt gave a characteristic cleavage pattern by the loss of the ethyl group of Ile at m/z 726.86 (calcd 726.79). The results show that the amino acid residues are distinguishable by the sidechain cleavage patterns. A similar fragmentation pattern was also shown in ESI-CID spectra (Figure 8). In this case, the fragment ions were observed in the divalent cation. 〈Ru〉-CO-Leu-OMe gave no fragment peak caused by the loss of two methyl groups around m/z 363 (Figure 6622 Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

8a). On the other hand, 〈Ru〉-CO-Ile-OMe indicates a clear fragment peak by the loss of an ethyl group at m/z 363.43 (Figure 8b). In ESI-MS, the fragmentation depended on the CID conditions (ionization energy, temperature, collision energy) to some degree. However, the ethyl group of the Ile residue cleaved easier than two methyl groups of the Leu residue similar to MALDI-LIFT analysis. The 〈Ru〉-CO-labeling method provides a characteristic sidechain cleavage pattern of amino acid residues in high sensitivity. Although the side-chain cleavage pattern depended on the fragmentation process by the MS/MS analytical method (MALDI-

Figure 9. MALDI-LIFT spectra of 〈Ru〉-Ile-Ser-bradykinin (a), its expanded spectrum in the range of m/z 600-850 (b), and expected fragment ions obtained from side-chain cleavage of N-terminal Ile residue (c).

Table 1. List of Fragment Ions of 〈Ru〉-CO-Xaa-OMe in Figure 5 Leu (m/z) found

calcd

M a1 + OH a1 a1 - CH3

815.13 771.02 755.05 742.05

815.22 771.19 755.19 741.81

d1 c0 a0

713.23 686.38 644.23

713.15 686.73 644.13

Ile (m/z)

M a1 + OH a1 a1 - CH3 d1 d1 - CH3 c0 a0

found

calcd

815.24 772.17 755.15 741.73 726.75 713.21 687.17 643.53

815.22 771.19 755.19 741.81 726.79 713.15 686.73 644.13

LIFT or ESI-CID), each case indicated similar side-chain cleavage patterns, which showed differences between amino acid residues. MALDI-LIFT-TOF/TOF Analysis of 〈Ru〉-CO-Ile-Ser-bradykinin. In the case of the long-chain peptide, we used Ile-Ser-

bradykinin to perform the N-terminal sequencing by the determination method of amino acid residues with 〈Ru〉-CO labeling. Ile-Ser-bradykinin consists of 11 amino acid residues (H-Ile-SerArg-Pro- Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) and has an N-terminal isoleucine residue. 〈Ru〉-CO-Ile-Ser-bradykinin was analyzed by MALDI-LIFT (Figure 9). 〈Ru〉-CO-Ile-Ser-bradykinin showed a characteristic fragment peak at m/z 726.83 by the loss of the ethyl group of the isoleucine residue. A complete series of an fragment ions was observed. The dn fragment ions formed by the cleavage of the Cβ-Cγ bond were also found. The amino acid residues can be characterized from an and dn fragment ions. From a0, the amino acid sequences are read successively to reach the C-terminus. The labeling method provides highly sensitive, rapid, and accurate N-terminal sequencing. CONCLUSION The 〈Ru〉-CO-labeling method enables us to characterize amino acid residues by the analysis of their side-chain cleavage using Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

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MALDI-LIFT-TOF/TOF-MS or ESI-CID-MS/MS. The cleavage patterns are peculiar to each amino acid residue and enable us to distinguish Leu/Ile isomers. The fragmentation pattern depended on a fragmentation process by the MS/MS analytical method to some degree. However, the labeling method displayed similar fragmentation patterns in MALDI-LIFT and ESI-CID spectra and also distinguished Leu/Ile isomers. In the case of the long-chain peptide, the labeling method allowed rapid, easy, and unambiguous N-terminal sequencing by the detection of an and dn fragment ions. Our methods are applicable for N-terminal amino acid sequencing of the digested peptides derived from unknown protein. We are now applying this method to unknown proteins of thermophilic bacteria.

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ACKNOWLEDGMENT A.I. expresses his special thanks for the center of excellence (21COE) program “Creation of Integrated EcoChemistry of Osaka University”. This work was supported in part National Project on Protein Structural and Function Analyses, Japan and Grant-in-Aid for Scientific Research (A) (15201043) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Received for review March 30, 2005. Accepted August 12, 2005. AC050534O