J. Mass Spectrom. Soc. Jpn. Vol. 50, No. 6, 2002 COMMUNICATION Hydrogen-Attachment Dissociation (HAD) The Characteristics of In-source Decay in Mass S

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1 J. Mass Spectrom. Soc. Jpn. Vol. 50, No. 6, 2002 COMMUNICATION Hydrogen-Attachment Dissociation (HAD) The Characteristics of In-source Decay in Mass Spectrometric Degradation Methods Hydrogen-Attachment Dissociation (HAD) MitsuD (Received August 23, 2002; Accepted October 30, 2002) In-source decay (ISD) combined with matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometer (TOF MS) has been described by comparing with conventional mass spectrometric degradation (MSD) methods such as collision-induced dissociation (CID) and post-source decay (PSD). The ISD characteristic is the formation of c- and (z 2)-ions originated from the N C a bond cleavage on the peptide backbone, while the CID and PSD processes are the CO NH bond cleavage which brings about b- andy-ion. Furthermore, the ISD processes occurring with 337 nm laser photon irradiation for peptide or protein proceed resulting in the formation of hyper-valent radical species via intermolecular hydrogen transfer between matrix and analyte molecules following the non-ergodic N C a bond cleavage. The non-ergodic N C a bond cleavage occurs in the MALDI ion source within nanosecond order, as an a-cleavage initiated with radical site at the carbonyl carbon. The MALDI-ISD method has been applied to three peptides and five proteins. 1. [M H] [M Na] [M nh] n (mass spectrometric degradation, MSD) 1) MSD (collisioninduced dissociation, CID) (postsource decay, PSD) MSD MSD MS MS Graduate School of Integrated Science, Yokohama City University (22 2 Seto, Kanazawa-ku, Yokohama , Japan) takayama@yokohama-cu.ac.jp 1 2 MSD MSD NMR X MS MSD MSD MSD MSD (matrix-assisted laser desorption/ionization, MALDI) NH C a H a hydrogen-attachment dissociation (HAD) 337

2 M. Takayama Fig. 1. Nomenclature of peptide fragment ions. 2. (Mass Spectrometric Degradation, MSD) MSD [M H] [M Na] CID CO NH y- b- a- d- v- w- (Fig. 1). CID 1 (E lab ) ev (E cm ) y- b- NH 3 H 2 O MSD (electron ionization, EI) (fast atom bombardment, FAB) MSD MSD 1 MSD 2 MSD MSD MS/MS CID skimmer CID MSD MSD Collision-induced dissociation (CID) High-energy CID Low-energy CID Skimmer CID Surface-induced dissociation (SID) Post-source decay (PSD) Blackbody infrared radiative dissociation (BIRD) Infrared multi-photon dissociation (IRMPD) Electron capture dissociation (ECD) CID, SID, PSD 338

3 The Characteristics of In-source Decay in Mass Spectrometric Degradation Methods Fig. 2. Fragmentation of molecular-related ions. (a)-1 closed shell protonated molecule [M H] is relatively stable and (a)-2 the open shell molecular ion M is subject to fragmentation initiated by radical. (b) Model for the hydrogen-attachment dissociation (HAD) of a stable protonated molecule. BIRD IRMPD (energy randomization) CID PSD 3,000 Da ECD [M nh] n H e 2.2 MSD in-source fragmentation in-source decay (ISD) EI M [M H] [M Na] M (Fig. 2(a)-2). ISD CID PSD MSD 339

4 M. Takayama Fig. 3. Internal amino-acid sequences are obtained by a series of c-ions appeared in MALDI-TOF mass spectrum of a protein. MSD MSD (electron ionization, EI) (fast atom bombardment, FAB) (plasma desorption, PD) (matrixassisted laser desorption/ionization, MALDI) EI M (Fig. 2(a)-2), FAB, PD, MALDI [M H] (Fig. 2(a)-1), (Fig. 2 (b)). MALDI MSD hydrogen-attachment dissociation (HAD) 3. Hydrogen-Attachment Dissociation (HAD) 3.1 MALDI-ISD HAD MALDI time-offlight mass spectrometry (TOF MS) ISD ISD 1995 Brown linear TOF MS in-source fragmentation 2) 1997 MALDI-TOF MS 3), 4) Brown ISD 5) MALDI-TOF MS ISD 6) 8) (hydrogen atom, H ) Fig. 4. In-source decay spectra of (a) a deuteriumlabeled dodecapeptide (M r 1,433.6) and (b) a non-labeled dodecapeptide (M r 1,428.6). The mass di#erence between c 6 product ions in (a) and (b) indicates that the c 6 product ion at m/z 773 in (a) is not formed via intramolecular hydrogen abstraction from the Ala (C a ) carbon. Likewise, the mass di#erence between the c 8 d 3 ion at m/z 946 in (a) and the c 8 ion at m/z 943 in (b) indicates that the c 8 product ion at m/z 943 in (b) is not formed via intramolecular hydrogen abstraction from the Gly8 (C a ) carbon. Therefore, the hydrogen required to form c products does not originate from the hydrogen on either the C a or C a carbon. (attachment) NH C a (dissociation) 9) 3.2 MALDI-ISD HAD N- c- 340

5 The Characteristics of In-source Decay in Mass Spectrometric Degradation Methods Fig. 5. Partial in-source decay spectra of (a) a deuterium dodecapeptide (M-d 26, M r 1,454.6) obtained with a deuterium matrix 2,5-DHB-d 3, and (b) a non-labeled dodecapeptide (M, M r 1,428.6) obtained with a 2,5-DHB matrix. In (a), the the product c 3 d 11 at m/z 355 must be formed by deuterium abstraction and deuteronation. (c) As the molar ratio of analyte to matrix is 1 : 7,000, both deuterium and deuteron originate from the matrix 2,5-DHB-d 3. Fig. 6. (a) X-ray crystallographic study and scanning probe atomic force microscopic study of the 2,5-DHB matrix with and without peptide incorporation into the matrix crystal suggest that the hydrophilic (100) surface of the matrix crystal is covered with peptide molecules. 9) (b) Intermolecular hydrogen bonding between a carbonyl oxygen on the peptide backbone and a phenolic hydrogen in the matrix may enable a hydrogen atom to move preferentially from the matrix to the backbone carbonyl oxygen immediately after photon absorption. Fig. 3 Fig. 3 ISD c- Fig. 1 c- NH C a 8) 1. NH C a. 2. NH C a 1 (Ala-d 3 ) (Gly-d 2 ) a b (Fig. 4). (2,5-DHB-d 3 ) c- (Fig. 5). 3.3 HAD a MALDI-TOF MS ISD a 9) 341

the image (b). Fig. 8. Schematic illustration for the mechanism of c-ion and (z 2)-ion formation.

6 M. Takayama Fig. 7. Scanning probe atomic force microscopic images of the 2,5-DHB matrix (a) without and with ACTH peptide incorporation into the matrix crystal. 9) After peptide incorporation the layered steps in the image (a) that form on the (100) surface of 2,5-DHB disappear like in the image (b). Fig. 8. Schematic illustration for the mechanism of c-ion and (z 2)-ion formation. 9) The formation of both ions can be explained by the a-cleavage of a transient hypervalent radical that formed via intermolecular hydrogen hydrogen transfer. Fig. 6 (AFM) (Fig. 7) 2,5-DHB X Fig. 6 10), 11) Fig. 8 hydrogen-attachment dissociation (HAD) 3.4 HAD electron capture dissociation (ECD) HAD a NH C a a c- (z 2)- (Fig. 9). NH C a ECD c- z- 12), 13) [M nh] n 1 342

7 The Characteristics of In-source Decay in Mass Spectrometric Degradation Methods HAD NH C a a ECD MALDI-ISD HAD a 12) 14) MSD CID MSD 3.5 MALDI-ISD energy-sudden desorption (ESD) MALDI ISD c- (z 2) Williams FABMS 15) Kenny 16) Biemann 17) LSIMS FABMS c- FABMS c- 18) (plasma desorption, PD) c- 19) NH C a c- FAB LSIMS PD MALDI energy-sudden desorption (ESD) 14) 3.6 ISD PSD In-source decay (ISD) post-source decay (PSD) source (ion source) ISD PSD ISD PSD (Fig. 10). ISD m i (i 1, 2, 3, ) [M H] MALDI-TOF Fig. 9. Hydrogen attachment dissociation (HAD) occurs via a-cleavage of a peptide radical. Fig. 10. Schematic illustration of reflectron time-offlight mass spectrometer equipped with matrixassisted laser desorption/ionization. In-source decay occurs within nano-s order in the ionization cell, while post-source decay occurs within micro-s order in the flight-tube. Fig. 11. (a) In-source decay spectrum and (b) post-source decay spectrum of substance P (RPKPQQFFGLM NH 2, M r 1,347.7). 343

8 M. Takayama PSD [M H] TOF MS [M H] m i (i 1, 2, 3, ) m i (i 1, 2, 3, ) [M H] m i 14) PSD [M H] m i m i m i ISD PSD Fig. 11 P (RPKPQQFFGLM-NH 2, M r 1,347.7) (a) ISD (b) PSD ISD 45 u c- a- Gly9 N- NH C a c 8 - Gly9 C- C a CO a 9 - MALDI-ISD 14) ISD 4.1 PSD a- (a 17)- PSD H 2 O 4. HAD DNA DNA DNA 10 MALDI-TOF MS HAD 4.1 HAD MALDI-ISD HAD MSD CID PSD NH C a a 4. [M H] [M H] M 5. N- NH C a 6. N- NH C a 7. NH C a 8. c- b- a- C- C a CO a C- y- (z 2)- 11. y- (z 2)- 12. ISD 2,5- (2,5-DHB) ISD ISD c- y- (z 2)- 15 u 344

9 The Characteristics of In-source Decay in Mass Spectrometric Degradation Methods Fig. 12. In-source decay spectrum of sulfonated tyrosine-containing peptide CCK-33 (M r 3,918.3). M r 10,000 m/z 2,000 5,000 ISD MALDI-ISD 2,5-DHB 0.1 TFA (50, v/v) 10,000 : 1 2,5-DHB X 2,5-DHB 9) 4.2 HAD (Lys, Arg, His) N- a-, b-, c- C- y- (z 2)- 7) C- N- 10a y 9-1. c- a- 45 u 2. (z 2)- y- 15 u HAD 20), 21) 8) Fig. 12 (Tyr27) (CCK-33) ISD 8) KAPSGRVSMIKNLQSLDPSHRISDRDY(SO 3 H)MGW- MDF NH 2 (M r 3,918.3) CCK-33 N- c- [M H] ( SO 3 ) m/z 3,840 c 26 - c HAD M r 10,000 MALDI-ISD a HAD Fig. 13 (M r 16,951) ISD m/ z 2,000 6,000 HAD c- a- (z 2)- 15 u y- 345

10 M. Takayama Fig. 13. In-source decay spectrum of horse hurt apomyoglobin (M r 16,951). Fig. 14. Partial in-source decay spectrum of horse hurt apomyoglobin (M r 16,951). Dot indicates a pair of (z 2)- and y-ions in the mass di#erence 15 u. Fig. 15. Partial in-source decay spectrum of horse hurt apomyoglobin (M r 16,951). Internal amino-acid sequence can be elucidated from the observed c-ions. m/z 2,000 4,000 c- 15 u (z 2)- y- (Fig. 14). m/z 4,000 6,000 c- (Fig. 15). c- Fig. 15 c 38 c T--L or I--E--Q or K F D--Q or K F Q or K H D Q ork T orv Web uk/fasta33/ Protein Data Bank T L E K F D K F K H L K--T C- 3 (L) (D) S/N S S ribonuclease A (M r 13,682), (M r 17,200), cucumber green mottle mosaic virus (CGMMV) (M r 17,305), c (M r 12,360) ISD Fig. 16 ribonuclease A (M r 13,682) c- m/z 1,000 3,000 c- 346

11 The Characteristics of In-source Decay in Mass Spectrometric Degradation Methods Fig. 16. Partial in-source decay spectrumof bovine ribonuclease A (M r 13,682). Fragment ions of c 26 or above were not observed due to the presence of a disulfide bond at Cys26. Fig. 17. Partial in-source decay spectrumof spermwhale apomyoglobin (M r 17,200). Fragment ion of c 36 was not observed due to the presence of Pro37. Fig. 18. Partial in-source decay spectrum of the coat protein of cucumber green mottle mosaic virus (M r 17,305). Fig. 19. In-source decay spectrumof equine cytochrome c (M r 12,360). Fragment ions of c 43 and c 44 were not observed due to the presence of Pro44 and Gly45. (M r 3,482.8) (Fig. 20). MALDI c- 5. MS MALDI-TOF MS ISD ISD a hydrogen-attachment dissociation (HAD) HAD MS CID, PSD, SID 347

12 M. Takayama Fig. 20. In-source decay spectrum of human glucagons (M r 3,482.8) and the several isotopic patterns for c-ion observed. MALDI-ISD 22) 30 N- c- HAD c- MATSUDA JHUPO 1996 MALDI-ISD MS 1) M. Takayama and A. Tsugita: Mass Spectrometry in Protein Study, in New Advances in Analytical Chemistry, ed. by Atta-ur-Rahman, Taylor & Francis, London (2002), pp ) R. S. Brown and J. J. Lennon, Anal. Chem., 67, (1995). 3) A. Tsugita, M. Kamo, K. Miyazaki, M. Takayama, T. Kawakami, R. Shen, and T. Nozawa, Electrophoresis, 19, (1998). 4) M. Takayama, T. Matsui, T. Sakai, and A. Tsugita, J. Biomol. Tech., 10, (1999). 5) M. Takayama and A. Tsugita, Int. J. Mass Spectrom., 181, L1 L6 (1998). 6) J. J. Lennon and K. A. Walsh, Protein Science, 6, (1997). 7) V. Katta, D. T. Chow, and M. F. Rohde, Anal. Chem., 70, (1998). 8) M. Takayama and A. Tsugita, Electrophoresis, 21, (2000). 9) M. Takayama, J. Am. Soc. Mass Spectrom., 12, (2001). 10) D. J. Harvey, Org. Mass Spectrom., 28, (1993). 11) I. Vidavsky, R. A. Chorush, P. Longevialle, and F. W. McLa#erty, J. Am. Chem. Soc., 116, (1994). 12) R. A. Zubarev, N. L. Kelleher, and F. W. McLa#erty, J. Am. Chem. Soc., 120, (1998). 13) R. A. Zubarev, N. A. Kruger, E. K. Fridriksson, M. A. Lewis, D. M. Horn, B. K. Carpenter, and F. W. Mc- La#erty, J. Am. Chem. Soc., 121, (1999). 14) M. Takayama, J. Am. Soc. Mass Spectrom., 12, (2001). 15) D. H. Williams, C. Bradley, G. Bojesen, S. Santiklarn, and L. C. E. Tayler, J. Am. Chem. Soc., 103, (1981). 16) P. T. M. Kenny, W.-H. Xu, K. Tachibana, and H. Naoki, in Proceedings of the 2 nd Japan China Joint Symposium on Mass Spectrometry (1987), pp ) K. Biemann, Biomed. Environ. Mass Spectrom., 16, (1988). 18) J. F. Mahoney, J. Perel, S. A. Ruatta, P. A. Martino, S. Husain, and T. D. Lee, Rapid Commun. Mass Spectrom., 5, (1991). 19) H. J. Vorst, M. W. E. M. van Tilborg, P. A. van Veelen, U. R. Tjaden, and J. van der Greef, Rapid Commun. Mass Spectrom., 4, (1990). 348

13 The Characteristics of In-source Decay in Mass Spectrometric Degradation Methods 20) J. J. Lennon and K. A. Walsh, Protein Science, 8, (1999). 21) T. Kinumi, H. Niwa, and H. Matsumoto, Anal. Biochem., 277, (2000). 22) J. Gao, A. Tsugita, M. Takayama, and L. Xu, Anal. Chem., 74, (2002). Keywords: MALDI, In-source decay, Mass spectrometric degradation, Hydrogen-attachment dissociation 349

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PowerPoint プレゼンテーション Mass Spectroscopy(MS) EI electron impact, electron impact ionization, electron bombardment ionization ( ) IUPAC electron ionization ( ) ( 31 (1997)) MS m/z( / ) 1 C 12.011 C 98.9% 13 C 1.1% m/z m z m/z