- Email: [email protected]
Proposed nomenclature for peptide ion fragmentation
G Model
ARTICLE IN PRESS
MASPEC-15461; No. of Pages 4
International Journal of Mass Spectrometry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Proposed nomenclature for peptide ion fragmentation夽 Ivan K. Chu a , Chi-Kit Siu b , Justin Kai-Chi Lau c,d , Wai Kit Tang b , Xiaoyan Mu a , Cheuk Kuen Lai a , Xinhua Guo e , Xian Wang f , Ning Li g , Yu Xia h , Xianglei Kong i , Han Bin Oh j , Victor Ryzhov k , Frantiˇsek Tureˇcek l , Alan C. Hopkinson c , K.W. Michael Siu c,d,∗ a
Department of Chemistry, The University of Hong Kong, Hong Kong, China Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China c Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3 d Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Avenue, Windsor, Ontario, Canada N9B 3P4 e College of Chemistry, Jilin University, Changchun, Jilin, China f College of Chemistry and Materials Science, South-Central University for Nationalities, Wuhan, Hubei, China g Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China h Department of Chemistry, Purdue University, IN, USA i State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, China j Department of Chemistry, Sogang University, Seoul, Republic of Korea k Department of Chemistry and Biochemistry, and Center for Biochemical and Biophysical Studies, Northern Illinois University, DeKalb, IL, USA l Department of Chemistry, University of Washington, Seattle, WA, USA b
a r t i c l e
i n f o
Article history: Received 7 March 2015 Received in revised form 29 June 2015 Accepted 20 July 2015 Available online xxx
a b s t r a c t The multitude of fragmentation techniques available to modern tandem mass spectrometry introduces diversity in the types of product ions. An all-explicit nomenclature system for the product ions of peptide fragmentation is herein proposed for the sake of clarity and unambiguity. All variables – the charge, radical and hydrogens gained or lost – associated with a given product ion are specified. © 2015 Published by Elsevier B.V.
Keywords: Nomenclature Peptide Dissociation Charge Radical
1. Introduction The fragmentation of peptide ions in the gas phase has piqued the interest of many a scientist [1–4]. In addition to providing a means to probe structure, these reactions constitute the basis for peptide identification either de novo or via a library search [4]. For ease of communication, nomenclature systems have been proposed to describe the product ions that can be formed. The first of such a system, proposed by Roepstorff and Fohlman [5], covers the cleavage of every single C␣ C, C N, and N C␣ bond (see Fig. 1, which
夽 Based in part on a presentation and discussion at the Mass Spectrometry and Proteomics Workshop 2014, December 13, 2014, University of Hong Kong, Hong Kong, China. ∗ Corresponding author at: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada. Tel.: +1 519 253 3000x3925. E-mail address: [email protected] (K.W.M. Siu).
is adapted from the original article) in a given protonated peptide (the “ionizing” proton is implicit in Fig. 1). The product ions are named A, B, and C ions, respectively, if the charge is retained on the N-terminal fragment; and X, Y, and Z ions, respectively, if it is retained on the C-terminal fragment. The subscript numbers indicate the residues involved, counting from the relevant terminal. Defining features of the Roepstorff–Fohlman system are that lettering is in the upper case; the ion charge is implicit (and always 1+); and any hydrogen “gained” is explicitly shown by the prime symbol. In Fig. 1c, the Y2 ion shown is a protonated dipeptide, which can conceptually be formed by adding two hydrogens to the Y2 fragment as designated in Fig. 1a. Nowadays, the Roepstorff–Fohlman notation has largely been superseded by the Biemann system [6], which features an expanded repertoire of fragment ions, including those that originate from the cleavage of side-chain groups (see Fig. 2, reproduced from the original work). The lettering for the fragment ions is now in italicized
http://dx.doi.org/10.1016/j.ijms.2015.07.021 1387-3806/© 2015 Published by Elsevier B.V.
Please cite this article in press as: I.K. Chu, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.07.021
G Model MASPEC-15461; No. of Pages 4
ARTICLE IN PRESS I.K. Chu et al. / International Journal of Mass Spectrometry xxx (2015) xxx–xxx
2
Fig. 1. Roepstorff and Fohlman nomenclature system for protonated peptide fragmentation. Adapted from Ref. [5] with permission.
Fig. 3. Hydrogen-rich radical peptide ion fragmentation. Reproduced from Ref. [3] with permission.
lower case, and the ion charge as well as hydrogens gained is implicit. The C-terminal product ion that results from cleavage of the amide C N bond is simply the yn ion – no plus sign and no prime symbol. The simplicity of the Biemann notation is perhaps an important contributor to its popularity and near universal adoption [4,7]. With the advent of electrospray ionization and the existence of multiply charged peptide ions [8], some product ions can now have more than one charge; for these ions, their Biemann notations
are almost always modified by addition of the charge explicitly, e.g., for a doubly charged y5 ion, the modified notation is y5 2+ [7]. In electron capture dissociation [9] and electron transfer dissociation [10], the possibility arises that radical cationic products are now possible. An example is shown in Fig. 3 (reproduced from the original [3]): cleavage of the N C␣ bond can give rise to either the c+ or the z•+ ion. Herein Tureˇcek and Julian [3] employed a format in which both the charge and the radical are explicit, while any hydrogens gained are implicit. It is noteworthy that the use
Fig. 2. Biemann nomenclature system for protonated peptide fragmentation. Reproduced from Ref. [6] with permission.
Please cite this article in press as: I.K. Chu, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.07.021
G Model MASPEC-15461; No. of Pages 4
ARTICLE IN PRESS I.K. Chu et al. / International Journal of Mass Spectrometry xxx (2015) xxx–xxx
3
Table 1 Conversion between the Biemann/Bowie notations and the proposed all-explicit nomenclature system. Cations Biemann b2 a2 y4 b18 2+ y13 2+ c2 a3 + 1
Anions Proposed [b2 ]+ [a2 ]+ [y4 + 2H]+ [b18 + H]2+ [y13 + 3H]2+ [c2 + 2H]+ [a3 + H]• +
Biemann/Roepstorff y1 b2 a2 c2
Proposed [y1 ]− [b2 − 2H]− [a2 ]− [c2 ]−
Bowie ␣  ’ ␥ ␦
Proposed [yn ]− [bn − 2H]− [bn + O]− [zn − 2H]− [cn ]−
implicit charge and hydrogen gained or lost in their negative electron transfer dissociation experiments. For the sake of clarity, we are proposing herein a comprehensive peptide fragmentation nomenclature system that avoids ambiguity in labeling the fragment ions observed in peptide dissociations. 2. Discussion
Fig. 4. Molecular radical peptide fragmentation. Material taken from Ref. [13] with permission.
of explicit charge and/or radical is not universal (see, e.g., [9,11]), which can cause confusion when new or unusual ions are involved. For molecular radical peptide fragmentation, the chemistry is arguably richer and there is a diversity of fragmentation products [2,12,13]. This is illustrated in Fig. 4 (material taken from [13]) which shows apparent cleavage of all three types of bonds – C␣ C, C N, and N C␣ . In addition, hydrogens can migrate from the Nterminal to the C-terminal products, and vice versa, which result in both hydrogen gains and losses. This complexity and diversity make explicitness mandatory for the sake of unambiguity. The system employed by Siu et al. [13] shows explicit charge, radical and hydrogens gained or lost. In the fragmentation of anionic peptides, Bowie et al. [14,15] have employed a drastically different nomenclature system. They used ␣- and -ions to represent the two possible product ions observed in cleavage of the C N bond, while ␥- and ␦-ions for those in the dissociation of the N C␣ bond. By contrast, Harrison [16,17] and Li et al. [18] favored using the Roepstorff–Fohlman (prime symbol to indicate the hydrogen gained and lost) and Biemann (lower case letters) notations to identify the product ions observed in the CID spectra of deprotonated peptides. For the anionic radical peptides, Chu and coworkers [19,20] employed a format resembling the molecular radical cations by showing explicit charge, radical, and hydrogen gained or lost in the fragment ions. However, Huzarska et al. [21] used the Biemann format to label the fragment ions with
Recognizing that the diversity of fragmentation techniques introduces a multitude of fragment types and fragmentation products, and recognizing that clarity and unambiguity are of paramount importance, we are recommending that all variables involved – charges, radicals and hydrogens gained or lost – need to be explicitly shown. To differentiate the proposed system from the Biemann system (which will no doubt be continually used by many involved in the fragmentation of protonated peptides and has served well for that chemistry type), all lettering will be in normal/regular font (no italics) and in lower case. To illustrate the proposed, all-explicit, notation, Table 1 shows some conversion between the Biemann/Bowie systems and the proposed system; square brackets are needed in the proposed system to make the notation clear when hydrogens are involved: Product ions from electron capture/transfer dissociation (socalled hydrogen-rich radical ions) are more varied due to the diversity in fragmentation chemistry. In our opinion, the proposed all-explicit system is required for clarity and unambiguity. For example, cm n+ ions described in the literature can formally arise by various processes. According to the proposed nomenclature system, a [cm + (n + 1)H]n+ even-electron ion depicts cleavage of the N C␣ bond of the (m + 1)th residue, accompanied by the addition of a hydrogen and n protons. Likewise, the cm − 1 ions frequently found in electron transfer dissociation spectra are denoted as [cm + nH]•n+ ions in the proposed system to capture their radical cation nature. By contrast, most zm n+ ions reported in the literature are [zm + nH]•n+ ions, and likewise the zm + 1 ions are denoted as [zm + (n + 1)H]n+ in the proposed system. Thus, if the fragment ion is identified, its stoichiometry can be explicitly described by the proposed symbol. Please see Fig. 4 and References [12,13] for examples of product ions generated from the fragmentation of radical peptides, e.g., [z2 − H]•+ and [b2 − H]•+ , that have no precedents in the Biemann notation. For these products, the only sensible notation is the allexplicit system. A somewhat more complicated situation arises when describing fragment ions formed by radical-induced loss of a side-chain, such as the C7 H6 O tyrosine or C9 H7 N tryptophan neutral fragments, which is typical of dissociations of hydrogen-deficient peptide radical ions. In this case, the backbone is formally conserved and the [a, b, c, x, y, z] system is not applicable; however, the affected residue has been modified, e.g., tyrosine is transformed into glycine with the radical at the ␣-carbon. We propose to use the
Please cite this article in press as: I.K. Chu, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.07.021
G Model MASPEC-15461; No. of Pages 4
ARTICLE IN PRESS I.K. Chu et al. / International Journal of Mass Spectrometry xxx (2015) xxx–xxx
4
Fig. 5. Molecular radical anionic peptide fragmentation. Material taken from Ref. [19] with permission. Product ions have been relabeled using the proposed all-explicit nomenclature system.
linear notation of the peptide sequence in which the known or presumed position of the radical modification is denoted. For example, the fragment ion formed by C7 H6 O loss from the YGAFL•+ radical cation is denoted as [G␣ • GAFL]+ ; the square brackets signify that the ion is a fragment, and G␣ • represents the glycine product with the radical at the ␣-carbon. If the radical moves to the -position on phenylalanine, the symbol for this product is [GGAF • L]+ , and likewise for other combinations of ␣ and  radical positions. It is recognized that specifying the complete sequence of a peptide fragment is not always convenient, especially when the peptide is long, and that the use of [M−106]•+ to signify loss of the tyrosine sidechain and [M−129]•+ to indicate loss of the tryptophan side-chain has had wide adoption [12,22–25]. The use of this shorthand notation is not discouraged as long as the identity of the residue involved and the location of the radical are clear. It should be noted that the proposed all-explicit system has already been adopted by some to describe fragments of hydrogendeficient peptide radical ions. For example, the [b2 − H]•+ notation was used for the fragmentation products of [C(S• )GG]+ (a sulfurcentered radical) [26] and [G␣ • SW]+ [27]. Similarly, Wee et al. [28] used G• XR and GX• R to indicate that the radical is located on the first and second residue, respectively; inclusion of a subscript to indicate the position of the radical (e.g., G␣ • XR and GX␣ • R) can further enhance the clarity, and is encouraged where appropriate. It is also noteworthy that this nomenclature system is applicable to the products of radical-driven peptide fragmentation mass spectrometry in which a radical initiator is conjugated to some part of the peptide [25,29,30]. We recommend adoption of the all-explicit system in describing the fragmentation of anionic peptides, which exhibits extensive product diversity and complexity, e.g., in the dissociation of the [M−2H]•− ion where M = RVYVHPL, the [x6 − 2H]− , [b5 − 2H]− , [z5 − 3H]•− and [z4 − H]•− ions were abundant (Fig. 5, material taken from Ref. [19]). References [1] B. Paizs, S. Suhai, Fragmentation pathways of protonated peptides, Mass Spectrom. Rev. 24 (2005) 508–548. [2] A.C. Hopkinson, Radical cations of amino acids and peptides: structures and stabilities, Mass Spectrom. Rev. 28 (2009) 655–671. [3] F. Tureˇcek, R.R. Julian, Peptide radicals and cation radicals in the gas phase, Chem. Rev. 113 (2013) 6691–6733.
[4] R. Aebersold, D.R. Goodlett, Mass spectrometry in proteomics, Chem. Rev. 101 (2001) 265–269. [5] P. Roepstorff, J. Fohlman, Proposal for a common nomenclature for sequence ions in mass spectra of peptides, Biomed. Mass Spectrom. 11 (1984) 601. [6] K. Biemann, Nomenclature for peptide fragment ions (positive ions), Methods Enzymol. 193 (1990) 886–887. [7] M.A. McFarland, C.L. Hendrickson, A.G. Marshall, Ion “threshing”: collisionally activated dissociation in an external octopole ion trap by oscillation of an axial electric potential gradient, Anal. Chem. 76 (2004) 1545–1549. [8] M. Mann, C.K. Meng, J.B. Fenn, Interpreting mass spectra of multiply charged ions, Anal. Chem. 61 (1989) 1702–1708. [9] R.A. Zubarev, N.L. Kelleher, F.W. McLafferty, Electron capture dissociation of multiply charged protein cations. A nonergodic process, J. Am. Chem. Soc. 120 (1998) 3265–3266. [10] J.E.P. Syka, J.J. Coon, M.J. Schroeder, J. Shabanowitz, D.F. Hunt, Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 9528–9533. [11] R. Mihalca, Y.E.M. van der Burgt, L.A. McDonnell, M. Duursma, I. Cerjak, A.J.R. Heck, R.M.A. Heeren, Combined infrared multiphoton dissociation and electron-capture dissociation using co-linear and overlapping beams in Fourier transform ion cyclotron resonance mass spectrometry, Rapid Commun. Mass Spectrom. 20 (2006) 1838–1844. [12] E. Bagheri-Majdi, Y. Ke, G. Orlova, I.K. Chu, A.C. Hopkinson, K.W.M. Siu, Coppermediated peptide radical ions in the gas phase, J. Phys. Chem. B 108 (2004) 11170–11181. [13] C.-K. Siu, Y. Ke, G. Orlova, A.C. Hopkinson, K.W.M. Siu, Dissociation of the N–C␣ bond and competitive formation of the [zn –H]• + and [cn + 2H]+ product ions in radical peptide ions containing tyrosine and tryptophan: the influence of proton affinities on product formation, J. Am. Soc. Mass Spectrom. 19 (2008) 1799–1807. [14] J.H. Bowie, C.S. Brinkworth, S. Dua, Collision-induced fragmentations of the [M−H]− parent anions of underivatized peptides: an aid to structure determination and some unusual negative ion cleavages, Mass Spectrom. Rev. 21 (2002) 87–107. [15] D. Bilusich, J.H. Bowie, Fragmentations of (M−H)− anions of underivatised peptides. Part 2: characteristic cleavages of Ser and Cys and of disulfides and other post-translational modifications, together with some unusual internal processes, Mass Spectrom. Rev. 28 (2009) 20–34. [16] A.G. Harrison, Sequence-specific fragmentation of deprotonated peptides containing H or alkyl side chains, J. Am. Soc. Mass Spectrom. 12 (2001) 1–13. [17] A.G. Harrison, Effect of phenylalanine on the fragmentation of deprotonated peptides, J. Am. Soc. Mass Spectrom. 13 (2002) 1242. [18] Z. Li, T. Yalcin, C.J. Cassady, C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus, J. Mass Spectrom. 41 (2006) 939–949. [19] C.N.W. Lam, I.K. Chu, Formation of anionic peptide radicals in vacuo, J. Am. Soc. Mass Spectrom. 17 (2006) 1249–1257. [20] J. Laskin, Z. Yang, C. Lam, I.K. Chu, Charge-remote fragmentation of odd-electron peptide ions, Anal. Chem. 79 (2007) 6607–6614. [21] M. Huzarska, I. Ugalde, D.A. Kaplan, R. Hartmer, M.L. Easterling, N.C. Polfer, Negative electron transfer dissociation of deprotonated phosphopeptide anions: choice of radical cation reagent and competition between electron and proton transfer, Anal. Chem. 82 (2010) 2873–2878. [22] I.K. Chu, C.F. Rodriquez, T.-C. Lau, A.C. Hopkinson, K.W.M. Siu, Molecular radical cations of oligopeptides, J. Phys. Chem. B 104 (2000) 3393–3397. [23] J. Laskin, Z. Yang, C.M.D. Ng, I.K. Chu, Fragmentation of ␣-radical cations of arginine-containing peptides, J. Am. Soc. Mass Spectrom. 21 (2010) 511–521. [24] H.B. Oh, B. Moon, Radical-driven peptide backbone dissociation tandem mass spectrometry, Mass Spectrom. Rev. 34 (2015) 116–132. [25] T. Ly, R.R. Julian, Tracking radical migration in large hydrogen deficient peptides with covalent labels: facile movement does not equal indiscriminate fragmentation, J. Am. Soc. Mass Spectrom. 20 (2009) 1148–1158. [26] A.K. Lam, V. Ryzhov, R.A.J. O’Hair, Mobile protons versus mobile radicals: gas-phase unimolecular chemistry of radical cations of cysteine-containing peptides, J. Am. Soc. Mass Spectrom. 21 (2010) 1296–1312. [27] Q. Quan, Q. Hao, T. Song, C.-K. Siu, I.K. Chu, Mechanistic investigation of phosphate ester bond cleavages of glycylphosphoserinyltryptophan radical cations under low-energy collision-induced dissociation, J. Am. Soc. Mass Spectrom. 24 (2013) 554–562. [28] S. Wee, R.A.J. O’Hair, W.D. McFadyen, Comparing the gas-phase fragmentation reactions of protonated and radical cations of the tripeptides GXR, Int. J. Mass Spectrom. 234 (2004) 101–122. [29] R. Hodyss, H.A. Cox, J.L. Beauchamp, Bioconjugates for tunable peptide fragmentation: free radical initiated peptide sequencing (FRIPS), J. Am. Chem. Soc. 127 (2005) 12436–12437. [30] M. Lee, M. Kang, B. Moon, H.B. Oh, Gas phase peptide sequencing by TEMPO mediated radical generation, Analyst 134 (2009) 1706–1712.
Please cite this article in press as: I.K. Chu, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.07.021
ARTICLE IN PRESS
MASPEC-15461; No. of Pages 4
International Journal of Mass Spectrometry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Proposed nomenclature for peptide ion fragmentation夽 Ivan K. Chu a , Chi-Kit Siu b , Justin Kai-Chi Lau c,d , Wai Kit Tang b , Xiaoyan Mu a , Cheuk Kuen Lai a , Xinhua Guo e , Xian Wang f , Ning Li g , Yu Xia h , Xianglei Kong i , Han Bin Oh j , Victor Ryzhov k , Frantiˇsek Tureˇcek l , Alan C. Hopkinson c , K.W. Michael Siu c,d,∗ a
Department of Chemistry, The University of Hong Kong, Hong Kong, China Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China c Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3 d Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Avenue, Windsor, Ontario, Canada N9B 3P4 e College of Chemistry, Jilin University, Changchun, Jilin, China f College of Chemistry and Materials Science, South-Central University for Nationalities, Wuhan, Hubei, China g Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China h Department of Chemistry, Purdue University, IN, USA i State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin, China j Department of Chemistry, Sogang University, Seoul, Republic of Korea k Department of Chemistry and Biochemistry, and Center for Biochemical and Biophysical Studies, Northern Illinois University, DeKalb, IL, USA l Department of Chemistry, University of Washington, Seattle, WA, USA b
a r t i c l e
i n f o
Article history: Received 7 March 2015 Received in revised form 29 June 2015 Accepted 20 July 2015 Available online xxx
a b s t r a c t The multitude of fragmentation techniques available to modern tandem mass spectrometry introduces diversity in the types of product ions. An all-explicit nomenclature system for the product ions of peptide fragmentation is herein proposed for the sake of clarity and unambiguity. All variables – the charge, radical and hydrogens gained or lost – associated with a given product ion are specified. © 2015 Published by Elsevier B.V.
Keywords: Nomenclature Peptide Dissociation Charge Radical
1. Introduction The fragmentation of peptide ions in the gas phase has piqued the interest of many a scientist [1–4]. In addition to providing a means to probe structure, these reactions constitute the basis for peptide identification either de novo or via a library search [4]. For ease of communication, nomenclature systems have been proposed to describe the product ions that can be formed. The first of such a system, proposed by Roepstorff and Fohlman [5], covers the cleavage of every single C␣ C, C N, and N C␣ bond (see Fig. 1, which
夽 Based in part on a presentation and discussion at the Mass Spectrometry and Proteomics Workshop 2014, December 13, 2014, University of Hong Kong, Hong Kong, China. ∗ Corresponding author at: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada. Tel.: +1 519 253 3000x3925. E-mail address: [email protected] (K.W.M. Siu).
is adapted from the original article) in a given protonated peptide (the “ionizing” proton is implicit in Fig. 1). The product ions are named A, B, and C ions, respectively, if the charge is retained on the N-terminal fragment; and X, Y, and Z ions, respectively, if it is retained on the C-terminal fragment. The subscript numbers indicate the residues involved, counting from the relevant terminal. Defining features of the Roepstorff–Fohlman system are that lettering is in the upper case; the ion charge is implicit (and always 1+); and any hydrogen “gained” is explicitly shown by the prime symbol. In Fig. 1c, the Y2 ion shown is a protonated dipeptide, which can conceptually be formed by adding two hydrogens to the Y2 fragment as designated in Fig. 1a. Nowadays, the Roepstorff–Fohlman notation has largely been superseded by the Biemann system [6], which features an expanded repertoire of fragment ions, including those that originate from the cleavage of side-chain groups (see Fig. 2, reproduced from the original work). The lettering for the fragment ions is now in italicized
http://dx.doi.org/10.1016/j.ijms.2015.07.021 1387-3806/© 2015 Published by Elsevier B.V.
Please cite this article in press as: I.K. Chu, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.07.021
G Model MASPEC-15461; No. of Pages 4
ARTICLE IN PRESS I.K. Chu et al. / International Journal of Mass Spectrometry xxx (2015) xxx–xxx
2
Fig. 1. Roepstorff and Fohlman nomenclature system for protonated peptide fragmentation. Adapted from Ref. [5] with permission.
Fig. 3. Hydrogen-rich radical peptide ion fragmentation. Reproduced from Ref. [3] with permission.
lower case, and the ion charge as well as hydrogens gained is implicit. The C-terminal product ion that results from cleavage of the amide C N bond is simply the yn ion – no plus sign and no prime symbol. The simplicity of the Biemann notation is perhaps an important contributor to its popularity and near universal adoption [4,7]. With the advent of electrospray ionization and the existence of multiply charged peptide ions [8], some product ions can now have more than one charge; for these ions, their Biemann notations
are almost always modified by addition of the charge explicitly, e.g., for a doubly charged y5 ion, the modified notation is y5 2+ [7]. In electron capture dissociation [9] and electron transfer dissociation [10], the possibility arises that radical cationic products are now possible. An example is shown in Fig. 3 (reproduced from the original [3]): cleavage of the N C␣ bond can give rise to either the c+ or the z•+ ion. Herein Tureˇcek and Julian [3] employed a format in which both the charge and the radical are explicit, while any hydrogens gained are implicit. It is noteworthy that the use
Fig. 2. Biemann nomenclature system for protonated peptide fragmentation. Reproduced from Ref. [6] with permission.
Please cite this article in press as: I.K. Chu, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.07.021
G Model MASPEC-15461; No. of Pages 4
ARTICLE IN PRESS I.K. Chu et al. / International Journal of Mass Spectrometry xxx (2015) xxx–xxx
3
Table 1 Conversion between the Biemann/Bowie notations and the proposed all-explicit nomenclature system. Cations Biemann b2 a2 y4 b18 2+ y13 2+ c2 a3 + 1
Anions Proposed [b2 ]+ [a2 ]+ [y4 + 2H]+ [b18 + H]2+ [y13 + 3H]2+ [c2 + 2H]+ [a3 + H]• +
Biemann/Roepstorff y1 b2 a2 c2
Proposed [y1 ]− [b2 − 2H]− [a2 ]− [c2 ]−
Bowie ␣  ’ ␥ ␦
Proposed [yn ]− [bn − 2H]− [bn + O]− [zn − 2H]− [cn ]−
implicit charge and hydrogen gained or lost in their negative electron transfer dissociation experiments. For the sake of clarity, we are proposing herein a comprehensive peptide fragmentation nomenclature system that avoids ambiguity in labeling the fragment ions observed in peptide dissociations. 2. Discussion
Fig. 4. Molecular radical peptide fragmentation. Material taken from Ref. [13] with permission.
of explicit charge and/or radical is not universal (see, e.g., [9,11]), which can cause confusion when new or unusual ions are involved. For molecular radical peptide fragmentation, the chemistry is arguably richer and there is a diversity of fragmentation products [2,12,13]. This is illustrated in Fig. 4 (material taken from [13]) which shows apparent cleavage of all three types of bonds – C␣ C, C N, and N C␣ . In addition, hydrogens can migrate from the Nterminal to the C-terminal products, and vice versa, which result in both hydrogen gains and losses. This complexity and diversity make explicitness mandatory for the sake of unambiguity. The system employed by Siu et al. [13] shows explicit charge, radical and hydrogens gained or lost. In the fragmentation of anionic peptides, Bowie et al. [14,15] have employed a drastically different nomenclature system. They used ␣- and -ions to represent the two possible product ions observed in cleavage of the C N bond, while ␥- and ␦-ions for those in the dissociation of the N C␣ bond. By contrast, Harrison [16,17] and Li et al. [18] favored using the Roepstorff–Fohlman (prime symbol to indicate the hydrogen gained and lost) and Biemann (lower case letters) notations to identify the product ions observed in the CID spectra of deprotonated peptides. For the anionic radical peptides, Chu and coworkers [19,20] employed a format resembling the molecular radical cations by showing explicit charge, radical, and hydrogen gained or lost in the fragment ions. However, Huzarska et al. [21] used the Biemann format to label the fragment ions with
Recognizing that the diversity of fragmentation techniques introduces a multitude of fragment types and fragmentation products, and recognizing that clarity and unambiguity are of paramount importance, we are recommending that all variables involved – charges, radicals and hydrogens gained or lost – need to be explicitly shown. To differentiate the proposed system from the Biemann system (which will no doubt be continually used by many involved in the fragmentation of protonated peptides and has served well for that chemistry type), all lettering will be in normal/regular font (no italics) and in lower case. To illustrate the proposed, all-explicit, notation, Table 1 shows some conversion between the Biemann/Bowie systems and the proposed system; square brackets are needed in the proposed system to make the notation clear when hydrogens are involved: Product ions from electron capture/transfer dissociation (socalled hydrogen-rich radical ions) are more varied due to the diversity in fragmentation chemistry. In our opinion, the proposed all-explicit system is required for clarity and unambiguity. For example, cm n+ ions described in the literature can formally arise by various processes. According to the proposed nomenclature system, a [cm + (n + 1)H]n+ even-electron ion depicts cleavage of the N C␣ bond of the (m + 1)th residue, accompanied by the addition of a hydrogen and n protons. Likewise, the cm − 1 ions frequently found in electron transfer dissociation spectra are denoted as [cm + nH]•n+ ions in the proposed system to capture their radical cation nature. By contrast, most zm n+ ions reported in the literature are [zm + nH]•n+ ions, and likewise the zm + 1 ions are denoted as [zm + (n + 1)H]n+ in the proposed system. Thus, if the fragment ion is identified, its stoichiometry can be explicitly described by the proposed symbol. Please see Fig. 4 and References [12,13] for examples of product ions generated from the fragmentation of radical peptides, e.g., [z2 − H]•+ and [b2 − H]•+ , that have no precedents in the Biemann notation. For these products, the only sensible notation is the allexplicit system. A somewhat more complicated situation arises when describing fragment ions formed by radical-induced loss of a side-chain, such as the C7 H6 O tyrosine or C9 H7 N tryptophan neutral fragments, which is typical of dissociations of hydrogen-deficient peptide radical ions. In this case, the backbone is formally conserved and the [a, b, c, x, y, z] system is not applicable; however, the affected residue has been modified, e.g., tyrosine is transformed into glycine with the radical at the ␣-carbon. We propose to use the
Please cite this article in press as: I.K. Chu, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.07.021
G Model MASPEC-15461; No. of Pages 4
ARTICLE IN PRESS I.K. Chu et al. / International Journal of Mass Spectrometry xxx (2015) xxx–xxx
4
Fig. 5. Molecular radical anionic peptide fragmentation. Material taken from Ref. [19] with permission. Product ions have been relabeled using the proposed all-explicit nomenclature system.
linear notation of the peptide sequence in which the known or presumed position of the radical modification is denoted. For example, the fragment ion formed by C7 H6 O loss from the YGAFL•+ radical cation is denoted as [G␣ • GAFL]+ ; the square brackets signify that the ion is a fragment, and G␣ • represents the glycine product with the radical at the ␣-carbon. If the radical moves to the -position on phenylalanine, the symbol for this product is [GGAF • L]+ , and likewise for other combinations of ␣ and  radical positions. It is recognized that specifying the complete sequence of a peptide fragment is not always convenient, especially when the peptide is long, and that the use of [M−106]•+ to signify loss of the tyrosine sidechain and [M−129]•+ to indicate loss of the tryptophan side-chain has had wide adoption [12,22–25]. The use of this shorthand notation is not discouraged as long as the identity of the residue involved and the location of the radical are clear. It should be noted that the proposed all-explicit system has already been adopted by some to describe fragments of hydrogendeficient peptide radical ions. For example, the [b2 − H]•+ notation was used for the fragmentation products of [C(S• )GG]+ (a sulfurcentered radical) [26] and [G␣ • SW]+ [27]. Similarly, Wee et al. [28] used G• XR and GX• R to indicate that the radical is located on the first and second residue, respectively; inclusion of a subscript to indicate the position of the radical (e.g., G␣ • XR and GX␣ • R) can further enhance the clarity, and is encouraged where appropriate. It is also noteworthy that this nomenclature system is applicable to the products of radical-driven peptide fragmentation mass spectrometry in which a radical initiator is conjugated to some part of the peptide [25,29,30]. We recommend adoption of the all-explicit system in describing the fragmentation of anionic peptides, which exhibits extensive product diversity and complexity, e.g., in the dissociation of the [M−2H]•− ion where M = RVYVHPL, the [x6 − 2H]− , [b5 − 2H]− , [z5 − 3H]•− and [z4 − H]•− ions were abundant (Fig. 5, material taken from Ref. [19]). References [1] B. Paizs, S. Suhai, Fragmentation pathways of protonated peptides, Mass Spectrom. Rev. 24 (2005) 508–548. [2] A.C. Hopkinson, Radical cations of amino acids and peptides: structures and stabilities, Mass Spectrom. Rev. 28 (2009) 655–671. [3] F. Tureˇcek, R.R. Julian, Peptide radicals and cation radicals in the gas phase, Chem. Rev. 113 (2013) 6691–6733.
[4] R. Aebersold, D.R. Goodlett, Mass spectrometry in proteomics, Chem. Rev. 101 (2001) 265–269. [5] P. Roepstorff, J. Fohlman, Proposal for a common nomenclature for sequence ions in mass spectra of peptides, Biomed. Mass Spectrom. 11 (1984) 601. [6] K. Biemann, Nomenclature for peptide fragment ions (positive ions), Methods Enzymol. 193 (1990) 886–887. [7] M.A. McFarland, C.L. Hendrickson, A.G. Marshall, Ion “threshing”: collisionally activated dissociation in an external octopole ion trap by oscillation of an axial electric potential gradient, Anal. Chem. 76 (2004) 1545–1549. [8] M. Mann, C.K. Meng, J.B. Fenn, Interpreting mass spectra of multiply charged ions, Anal. Chem. 61 (1989) 1702–1708. [9] R.A. Zubarev, N.L. Kelleher, F.W. McLafferty, Electron capture dissociation of multiply charged protein cations. A nonergodic process, J. Am. Chem. Soc. 120 (1998) 3265–3266. [10] J.E.P. Syka, J.J. Coon, M.J. Schroeder, J. Shabanowitz, D.F. Hunt, Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 9528–9533. [11] R. Mihalca, Y.E.M. van der Burgt, L.A. McDonnell, M. Duursma, I. Cerjak, A.J.R. Heck, R.M.A. Heeren, Combined infrared multiphoton dissociation and electron-capture dissociation using co-linear and overlapping beams in Fourier transform ion cyclotron resonance mass spectrometry, Rapid Commun. Mass Spectrom. 20 (2006) 1838–1844. [12] E. Bagheri-Majdi, Y. Ke, G. Orlova, I.K. Chu, A.C. Hopkinson, K.W.M. Siu, Coppermediated peptide radical ions in the gas phase, J. Phys. Chem. B 108 (2004) 11170–11181. [13] C.-K. Siu, Y. Ke, G. Orlova, A.C. Hopkinson, K.W.M. Siu, Dissociation of the N–C␣ bond and competitive formation of the [zn –H]• + and [cn + 2H]+ product ions in radical peptide ions containing tyrosine and tryptophan: the influence of proton affinities on product formation, J. Am. Soc. Mass Spectrom. 19 (2008) 1799–1807. [14] J.H. Bowie, C.S. Brinkworth, S. Dua, Collision-induced fragmentations of the [M−H]− parent anions of underivatized peptides: an aid to structure determination and some unusual negative ion cleavages, Mass Spectrom. Rev. 21 (2002) 87–107. [15] D. Bilusich, J.H. Bowie, Fragmentations of (M−H)− anions of underivatised peptides. Part 2: characteristic cleavages of Ser and Cys and of disulfides and other post-translational modifications, together with some unusual internal processes, Mass Spectrom. Rev. 28 (2009) 20–34. [16] A.G. Harrison, Sequence-specific fragmentation of deprotonated peptides containing H or alkyl side chains, J. Am. Soc. Mass Spectrom. 12 (2001) 1–13. [17] A.G. Harrison, Effect of phenylalanine on the fragmentation of deprotonated peptides, J. Am. Soc. Mass Spectrom. 13 (2002) 1242. [18] Z. Li, T. Yalcin, C.J. Cassady, C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus, J. Mass Spectrom. 41 (2006) 939–949. [19] C.N.W. Lam, I.K. Chu, Formation of anionic peptide radicals in vacuo, J. Am. Soc. Mass Spectrom. 17 (2006) 1249–1257. [20] J. Laskin, Z. Yang, C. Lam, I.K. Chu, Charge-remote fragmentation of odd-electron peptide ions, Anal. Chem. 79 (2007) 6607–6614. [21] M. Huzarska, I. Ugalde, D.A. Kaplan, R. Hartmer, M.L. Easterling, N.C. Polfer, Negative electron transfer dissociation of deprotonated phosphopeptide anions: choice of radical cation reagent and competition between electron and proton transfer, Anal. Chem. 82 (2010) 2873–2878. [22] I.K. Chu, C.F. Rodriquez, T.-C. Lau, A.C. Hopkinson, K.W.M. Siu, Molecular radical cations of oligopeptides, J. Phys. Chem. B 104 (2000) 3393–3397. [23] J. Laskin, Z. Yang, C.M.D. Ng, I.K. Chu, Fragmentation of ␣-radical cations of arginine-containing peptides, J. Am. Soc. Mass Spectrom. 21 (2010) 511–521. [24] H.B. Oh, B. Moon, Radical-driven peptide backbone dissociation tandem mass spectrometry, Mass Spectrom. Rev. 34 (2015) 116–132. [25] T. Ly, R.R. Julian, Tracking radical migration in large hydrogen deficient peptides with covalent labels: facile movement does not equal indiscriminate fragmentation, J. Am. Soc. Mass Spectrom. 20 (2009) 1148–1158. [26] A.K. Lam, V. Ryzhov, R.A.J. O’Hair, Mobile protons versus mobile radicals: gas-phase unimolecular chemistry of radical cations of cysteine-containing peptides, J. Am. Soc. Mass Spectrom. 21 (2010) 1296–1312. [27] Q. Quan, Q. Hao, T. Song, C.-K. Siu, I.K. Chu, Mechanistic investigation of phosphate ester bond cleavages of glycylphosphoserinyltryptophan radical cations under low-energy collision-induced dissociation, J. Am. Soc. Mass Spectrom. 24 (2013) 554–562. [28] S. Wee, R.A.J. O’Hair, W.D. McFadyen, Comparing the gas-phase fragmentation reactions of protonated and radical cations of the tripeptides GXR, Int. J. Mass Spectrom. 234 (2004) 101–122. [29] R. Hodyss, H.A. Cox, J.L. Beauchamp, Bioconjugates for tunable peptide fragmentation: free radical initiated peptide sequencing (FRIPS), J. Am. Chem. Soc. 127 (2005) 12436–12437. [30] M. Lee, M. Kang, B. Moon, H.B. Oh, Gas phase peptide sequencing by TEMPO mediated radical generation, Analyst 134 (2009) 1706–1712.
Please cite this article in press as: I.K. Chu, et al., Int. J. Mass Spectrom. (2015), http://dx.doi.org/10.1016/j.ijms.2015.07.021