Advanced
Charge-Directed Peptide Backbone Dissociations of <italic>o</italic>-TEMPO-Bz-C(O)-Peptides
Charge-Directed Peptide Backbone Dissociations of o-TEMPO-Bz-C(O)-Peptides
Mass Spectrometry Letters. 2013. Dec, 4(4): 71-74
Copyright ©2013, Korean Society Mass Spectrometry
All MS Letters content is Open Access, meaning it is accessible online to everyone, without fee and authors’ permission. All MS Letters content is published and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org /licenses/by/3.0/). Under this license, authors reserve the copyright for their content; however, they permit anyone to unrestrictedly use, distribute, and reproduce the content in any medium as far as the original authors and source are cited. For any reuse, redistribution, or reproduction of a work, users must clarify the license terms under which the work was produced.
  • Received : December 01, 2013
  • Accepted : December 02, 2013
  • Published : December 30, 2013
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Aeran Jeon
Department of Chemistry, Sogang University, Seoul 121-742, Korea
Ji Hye Lee
Department of Chemistry, Sogang University, Seoul 121-742, Korea
Hyuk Su Kwon
Department of Chemistry, Sogang University, Seoul 121-742, Korea
Hyung Soon Park
Diatech Korea Co. Ltd., Seoul 138-826, Korea
Bong Jin Moon
Department of Chemistry, Sogang University, Seoul 121-742, Korea
Han Bin Oh
Department of Chemistry, Sogang University, Seoul 121-742, Korea
hanbinoh@sogang.ac.kr

Abstract
In the present study, we report that the charge-directed (assisted) peptide dissociation products, such as b- and y- type pep-tide backbone fragments, were the major products in MS/MS and MS 3 applications of some o -TEMPO-Bz-C(O)-peptide ions, while radical-driven dissociation products, such as a/x and c/z -type fragments, were previously shown to be the major products in the free radical initiated peptide sequencing mass spectrometry (FRIPS MS). Those o -TEMPO-Bz-C(O)-peptides share a common feature in their sequences, that is, the peptides do not include an arginine residue that has the highest proton affinity among free amino acids. The appearance of b- and y- type fragments as major products in FRIPS MS can be understood in terms of the so-called “mobile-proton model”. When the proton is highly mobilized by the absence of arginine, the chare-directed peptide dissociation pathways appear to be more competitive than the radical-driven dissociation pathways, in our FRIPS experiments.
Keywords
Introduction
Since the discovery of electron capture dissociation (ECD) in 1998, radical-based peptide/protein tandem mass spectrometry has been the area of active research due to its powerful peptide dissociation capability and its unique opportunity for characterization of post-translational modifications. 1 - 14 In ECD, generation of a radical site on the peptide/protein backbone has been shown to be the key step in the ensuing peptide/protein dissociation. 1 , 3 , 5 Later, it was shown that a radical site could be generated on the peptide backbone through electron transfer from the negatively-charged anion to the protonated peptide cation; electron transfer dissociation (ETD). 15
These findings have led to the development of a variety of radical-based peptide/protein tandem mass spectrometry methods. For example, low-energy collision activation of ternary metal complexes with peptide and auxiliary ligands was shown to generate peptide radical ions. 16 - 19 A radical site could also be generated site-specifically on the phenol ring through UV irradiation of a photolabile radical precursor. 20 Another interesting approach to note is to conjugate a free radical initiator at the primary amine group of the lysine side chain or N-terminus of peptide; the so-called Free Radical 21 - 25 Initiated Peptide Sequencing (FRIPS) approach.Collisional activation can rupture the bond between the free radical initiator and the peptide, and as a result, creates a radical site. The generated radical site was found to result in unique peptide/protein backbone fragmentations upon another collisional activation application. Unlike the routine collisional activation dissociation (CAD), in these radical-based tandem mass spectrometry method, a- , c- , x- , and z- type fragment ions (refer to Scheme 1) have been observed to be the major fragments and side-chain loss occurs frequently. 22 , 23 In addition, the stability of the generated radical site and the transfer of the radical site have also been extensively studied to deepen the understanding of the radical-driven peptide
1-1
Lager Image
Scheme 1.
backbone dissociation mass spectrometry.
Our group introduced the conjugation of o -TEMPO-benzyl-NHS (ortho-TEMPO-methyl benzoic acid N-hydroxysuccinimide; TEMPO = 2,2,6,6-tetramethylpiperi-dine-1-oxyl) with the N-terminus of peptides, as a FRIPS approach. 23 - 25 Thermal energy provided through collisional activation leads to the generation of the benzylic peptide radical ions, i.e ., •-CH 2 -Bz-CO-Peptide. This radical generation step is facilitated and driven by the extraordinary thermodynamic stability of the TEMPO• radical. The subsequent application of collisional activation to the peptide radical ions results in extensive peptide backbone dissociations, particularly, a- , c- , x- , and z- type fragment ions, and side chain losses. Recently, our group reported that this two-step process can proceed in a single step application of CAD when applied in negative-ion mode. 25
Interestingly, in contrast to this general characteristic radical-driven peptide backbone dissociation behavior of the o -TEMPO-Bz-C(O)-peptide, it has been found that charge-driven peptide backbone dissociation products, such as b- and y- type fragments, are the major fragments for some peptides. In the present study, we have sought to find out which peptides show such abnormal fragmentation behavior and on what basis we can understand such interesting findings.
Experiments
The experimental procedure was previously detailed. 23 - 25 In brief, the peptides of interest was dissolved in a 0.1 M NaHCO 3 buffer solution and then subjected to conjugation with o -TEMPO-Bz-NHS in DMSO by mixing. The o -TEMPO-Bz-conjugated peptides were purified prior to tandem mass spectrometry application, using the reversed-phase solid phase extraction (SPE, Ultra-micro spin column C-18, Harvard Bioscience, Holliston, MA, USA) protocol. Mass spectrometry experiments were carried out on a linear ion-trap mass spectrometer (LTQ XL, Thermo Scientific, San Jose, CA, USA) in positive-ion mode. The conjugated peptides dissolved in a solution of water/methanol/acetic acid (v/v/v 49:49:2) was introduced into the mass spectrometer using directly-infused electrospray ionization (ESI) source at a flow rate of ~3 μL/min. The conjugated peptide ions were isolated and then subjected to tandem mass spectrometry analysis. The ESI mass spectra were obtained by averaging 50-100 scanned spectra. The three peptides (ALILTLVS, SNNFGAILSS, and VQGEESNDK) were purchased from Bachem (Budendorf, Switzerland) and used without further purification.
Results and Discussion
- Abnormal FRIPS behavior
Figure 1 a shows an MS/MS spectrum obtained at the normalized collision energy (NCE) of 24.5 (arbitrary unit) for o -TEMPO-Bz-C(O)-ALILTLVS singly protonated peptide. To our surprise, a large number of peptide backbone dissociation products are observed, in contrast to the previously-reported cases
Lager Image
(a) MS/MS and (b) MS3 mass spectra of o-TEMPOBz-C(O)-ALILTLVS singly protonated peptide. The subscript “R” at the left-side of M, e.g., (RM + H)+, denotes that o-TEMPO-Bz-C(O)- is conjugated to the N-terminus of the peptides. *: peak not assigned.
in which the generation of the benzylic peptide radical ions (•CH 2 -Bz-CO-Peptide) was a dominant process. 23 , 24 Analysis of the fragments in the MS/MS spectrum indicates that b- and y- type ions are the major products. For example, rb7 + at m/z 840.1 is the most abundant fragment, and rb5 + , rb6 + , y5 + , y6 + , and y7 + are also observed in significant abundance. Here, the subscript ‘ r ’ at the left-side of b ion, e.g ., rb7 + , indicates that the fragment contains the •CH 2 -Bz-CO- at the N-terminus of the peptide. Even at the lower NCE, e.g ., NCE = 19.0, the characteristic fragmentation pattern was almost similar to that at NCE = 24.5; that is, b- and y- type fragments were still the major products. Although the benzylic peptide radical ions, •CH 2 -Bz-CO-Peptide, ( r M + H) +• at m/z 946.1, were generated, its abundance was relatively low.
The subsequent collisional activation was applied to the benzylic peptide radical ions, and its resulting spectrum is shown in Figure 1 b. The obtained MS 3 spectrum is similar to the MS/MS spectrum ( Figure 1 a) in that b- and y- type fragments are the major products. In both spectra, some a- , x- and c- type fragments were observed, but as minor products; e.g ., x6 + , x7 + , ra7 + , and rc7 + .
The above-mentioned MS/MS and MS 3 results are abnormal in two respects. First, at MS/MS, the cleavage of the benzylic carbon and the TEMPO oxygen, which generates •CH 2 -Bz-CO-Peptide radical ions, did not occur predominantly. Second, upon MS/MS and MS 3 application, b- and y- type fragments appeared as major products. The b- and y- type fragments are known to be generated through a charge 18 , 26 , 27 directed (or assisted) peptide dissociation pathway. A radical-driven peptide backbone dissociation pathway that leads to the generation of a- , c- , x- , and z- type fragment ions does not seem to be the dominant fragmentation pathway for the peptide with the sequence of ALILTLVS.
Lager Image
(a) MS/MS and (b) MS3 mass spectra of o-TEMPO-Bz-C(O)-SNNFGAILSS singly protonated peptide. The subscript “R” at the left-side of M, e.g., (RM + H)+, denotes that o-TEMPO-Bz-C(O)-is conjugated to the N-terminus of the peptides. *: peak not assigned.
Lager Image
(a) MS/MS and (b) MS3 mass spectra of o-TEMPOBz-C(O)-VQGEESNDK doubly protonated peptide. The subscript “R” at the left-side of M, e.g., (RM + 2H)2+, denotes that o-TEMPO-Bz-C(O)- is conjugated to the N-terminus of the peptides. *: peak not assigned. Figure (b) is modified from Figure 4b in Analyst, 2009, 134, 1710 and reproduced with permission from the Royal Society of Chemistry, copyright 2009.
- Other peptides
Figures 2 and 3 show the MS/MS and MS 3 spectra for o -TEMPO-Bz-C(O)-SNNFGAILSS and o -TEMPO-Bz-C(O)VQGEESNDK, respectively. Both of the conjugated peptides show the abnormal FRIPS behaviour, which is similar to the case of ALILTLVS. Upon MS/MS application, these two conjugated peptides yielded a large number of b- and y- type fragments (see Figure 2 a and 3 a), which is a stark contrast to the normal FRIPS MS/MS in which • CH 2 -Bz-CO-peptide was an exclusive product. To further examine the dissociation behaviour of o -TEMPO-Bz-C(O)-peptides, the extra collisional activation was applied to the •CH 2 -Bz-CO-peptides that were generated in low abundance in the MS/MS experiments. Like the case of ALILTLVS, the two peptides yielded a large number of b- and y- type fragments (see Figure 2 b and 3 b).
- Mobile-proton model
It is quite exceptional that these three peptides (ALILTLVS, SNNFGAILSS, and VQGEESNDK) show the MS/MS and MS 3 fragmentation behaviour quite different from those of the previously-reported peptides. 21 , 23 - 25 A hint why these conjugated peptides show abnormal FRIPS dissociation behaviour may be found in the fact that these three peptides do not include an arginine (R) residue, which is known to have the highest proton affinity among twenty free amino acids, 28 in their sequences. Although not shown here, many other peptides without the arginine residue in sequence repeatedly showed the identical fragmentation behaviour, yielding b- and y- type ions as major products.
In other radical-based peptide dissociation methods, such as the low-energy collision activation of ternary metal complexes with peptide and auxiliary ligands and the UV photolysis of iodine-substituted radical precursor, it was clearly demonstrated that only the peptide radical ions containing an arginine residue yielded radical-driven dissociation products, such as a / x and c/z -type backbone fragments and side chain losses. 20 , 29 For those without an arginine, charge-directed peptide backbone dissociations tended to be dominant.
These observations can be understood in terms of the so-called “ mobile proton model ”. 30 When the arginine with the highest proton affinity is absent, a proton becomes so mobile that the charge-assisted peptide dissociation process is highly catalysed by the proton at the site where the proton is attached. In particular, the formation of oxazolone-type b ion was evidenced by many experimental and theoretical studies, though a six-membered diketopiperazine-type fragment was also an arguable candidate for the b ion structure. 26 , 27 , 31 In FRIPS MS experiments, the radical-driven peptide dissociation is generally in competition with the charge-assisted peptide dissociations. For the peptides with an arginine residue, the radical-driven peptide dissociation pathways, leading to a / x and c / z -type backbone fragments, are dominant pathways since the mobility of a proton is limited by the presence of arginine. However, when the most basic arginine does not exist in sequence, the charge-directed peptide dissociation process appears to be more competitive than the radical-driven process.
To summarize, we have here demonstrated that the charge-directed peptide dissociations, which yielded b- and y- type fragments, can be a dominant dissociation pathway when an arginine is absent in sequence. In order to develop FRIPS MS as a useful proteomics tool, it is necessary to consider this finding in the interpretation of the tandem mass spectra, and furthermore it may be needed to develop a method that can direct the peptide dissociations more favourably into the radical-driven dissociation pathways.
Acknowledgements
This research was supported by a grant from the Emerging Industrial Technology Development Program funded by the Ministry of Trade, Industry&Energy of Korea (10044352).
References
Zubarev R. A. , Kelleher N. L. , McLafferty F. W. 1998 J. Am. Chem. Soc. 120 3265 -    DOI : 10.1021/ja973478k
Oh H. B. , Breuker K. , Sze S. K. , Ying G. , Carpenter B. K. , McLafferty F. W. 2002 Proc. Natl. Acad. Sci. USA 99 15863 -    DOI : 10.1073/pnas.212643599
Zubarev R. A 2003 Mass Spectrom. Rev. 22 57 -    DOI : 10.1002/mas.10042
Leymarie N. , Costello C. E. , O’Connor P. B. 2003 J. Am. Chem. Soc. 125 8949 -    DOI : 10.1021/ja028831n
Cooper H. J. , Hakansson K. , Marshall A. G. 2005 Mass Spectrom. Rev. 24 201 -    DOI : 10.1002/mas.20014
Fung Y. M. E. , Chan T. W. D. 2005 J. Am. Soc. Mass Spectrom. 16 1523 -    DOI : 10.1016/j.jasms.2005.05.001
Oh H. B. , McLafferty F. W. 2006 Bull. Korean Chem. Soc. 27 389 -    DOI : 10.5012/bkcs.2006.27.3.389
Lee, S. Y. , Han S. Y. , Lee T. G. , Lee D. H. , Chung G. S. , Oh H. B. 2006 J. Am. Soc. Mass Spectrom. 17 536 -    DOI : 10.1016/j.jasms.2005.12.004
Yim Y. H. , Kim B. J. , Ahn S. H. , So H. Y. , Lee S. Y. , Oh H. B. 2006 Rapid Commun Mass Spectrom. 20 1918 -    DOI : 10.1002/rcm.2533
Lee S. Y. , Chung G. S. , Kim J. D. , Oh H. B. 2006 Rapid Commun Mass Spectrom. 20 3167 -    DOI : 10.1002/rcm.2708
Chen X. , Tureeek F. 2006 J. Am. Chem. Soc. 128 12520 -    DOI : 10.1021/ja063676o
Lee S. Y. , Park S. J. , Ahn S. H. , Oh H. B. 2009 Int. J. Mass Spectrom. 279 47 -    DOI : 10.1016/j.ijms.2008.10.008
Hamidane H. B. , Chiappe D. , Hartmer R. , Vorobyev A. , Moniatte M. , Tsybin Y. O. 2009 J. Am. Soc. Mass Spectrom. 20 567 -    DOI : 10.1016/j.jasms.2008.11.016
Kaczorowska M. A. , Hotze A. C. G. , Hannon M. J. , Cooper H. J. 2010 J. Am. Soc. Mass Spectrom. 21 300 -    DOI : 10.1016/j.jasms.2009.10.018
Syka J. E. , Coon J. J. , Schroeder M. J. , Shabanowitz J. , Hunt D. F. 2004 Proc. Natl. Acad. Sci. USA 101 9528 -    DOI : 10.1073/pnas.0402700101
Chu I. K. , Rodriguez C. F. , Lau T. C. , Hopkinson A. C. , Siu K. W. M. 2000 J. Phys. Chem. B 104 3393 -    DOI : 10.1021/jp994487d
Barlow C. K. , McFadyen, W. D. , O’Hair R. A. J. 2005 J. Am. Chem. Soc. 127 6109 -    DOI : 10.1021/ja043088f
Chu I. K. , Zhao J. , Xu M. , Siu S. O. , Hopkinson A. C. , Siu K. W. M. 2008 J. Am. Chem. Soc. 130 7862 -    DOI : 10.1021/ja801108j
Chu I. K. , Laskin J. 2011 Eur. J. Mass Spectrom. 17 543 -    DOI : 10.1255/ejms.1156
Sun Q. , Nelson H. , Ly T. , Stoltz B. M. , Julian R. R. 2009 J. Proteome Res. 8 958 -    DOI : 10.1021/pr800592t
Masterson D. S. , Yin H. , Chacon A. , Hachey D. L. , Norris J. L. , Porter N. A. 2004 J. Am. Chem. Soc. 126 720 -    DOI : 10.1021/ja038615u
Hodyss R. , Cox H. A. , Beauchamp J. L. 2005 J. Am. Chem. Soc. 127 12436 -    DOI : 10.1021/ja052042z
Lee M. H. , Kang M. H. , Moon B. J. , Oh H. B. 2009 Analyst 134 1706 -    DOI : 10.1039/b904115j
Lee M. H. , Lee Y. J. , Kang M. H. , Park H. Y. , Seong Y. M. , Sung B. J. , Moon B. J. , Oh H. B. 2011 J. Mass Spectrom. 46 830 -    DOI : 10.1002/jms.1955
Lee J. H. , Park H. Y. , Kwon H. S. , Kwon K. M. , Jeon A. R. , Kim H. I. , Sung B. J. , Moon B. J. , Oh H. B. 2013 Anal. Chem. 85 7044 -    DOI : 10.1021/ac303517h
Yalcin T. , Khouw C. , Csizmadia I. G. , Peterson M. R. , Harrison A. G. 1995 J. Am. Soc. Mass Spectrom. 6 1165 -    DOI : 10.1016/1044-0305(95)00569-2
Paizs B. , Suhai S. 2001 Rapid Commun. Mass Spectrom. 15 2307 -    DOI : 10.1002/rcm.507
Harrison A. G. 1997 Mass Spectrom. Rev. 16 201 -    DOI : 10.1002/(SICI)1098-2787(1997)16:4<201::AID-MAS3>3.0.CO;2-L
Laskin J. , Yang Z. , Lam C. , Chu I. K. 2007 Anal. Chem. 79 6607 -    DOI : 10.1021/ac070777b
Dongre A. R. , Jones J. L. , Somogyi A. , Wysocki V. H. 1996 J. Am. Chem. Soc. 118 8365 -    DOI : 10.1021/ja9542193
Cordero M. M. , Houser J. J. , Wesdemiotis C. 1993 Anal. Chem. 65 1594 -    DOI : 10.1021/ac00059a019