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Cite this: Chem. Commun., 2014, 50, 198 Received 19th August 2013, Accepted 15th October 2013
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Unusual ECD fragmentation attributed to gas-phase helix formation in a conformationally dynamic peptide† Jason M. D. Kalapothakis,a Yana Berezovskaya,a Cleidiane G. Zampronio,b Peter A. Faull,a Perdita E. Barran*a and Helen J. Cooper*b
DOI: 10.1039/c3cc46356g www.rsc.org/chemcomm
The helix-forming character of a model decapeptide, L4PL4K, is determined in the absence of solvent using ion mobility mass spectrometry, electron capture dissociation and molecular mechanics simulations. Unusual ECD fragmentation patterns dominated by b ions are attributed to helix formation upon electron capture and as a signature of conformational dynamics.
In recent years there has been an increase in the use of gas phase methods to determine higher order structures of biological molecular systems.1–3 Much of this work requires biological macromolecules to maintain their structure over short (> ms) experimental time scales; however this premise suffers in those cases where a system possesses fast conformational dynamics. In this work a simple model decapeptide, L4PL4K, is examined using ion mobility mass spectrometry (IM-MS), electron capture dissociation (ECD) and molecular mechanics simulations in order to delineate the effects of helix forming propensity on fragmentation behaviour. Following ECD (see ESI† for experimental details), the most notable feature of L4PL4K ECD fragmentation is the prominence of CID-like fragments, w fragments and H loss. Typically ECD yields c0 and z type fragments with cleavage along the N–Ca bond4,5 (Fig. 1A); however, L4PL4K constructs exhibit ECD spectra in which c and z fragment ions are relatively sparse, whereas b, y ion pairs as well as w ions (thought to result from higher-energy dissociation processes6) are most persistent (Fig. 1B–D and Fig. S1, ESI†). Side chain losses are prominent across all three constructs, with w6–10 and w5–4 being present in all ECD spectra of [M + 2H]2+ ions. These fragments are detected even in cases where the corresponding z ions are absent, as they are for the free termini and acetyl-free acid constructs w5, w7 and w8. Other ubiquitous fragments across all three constructs are z4, b2–5, b9 and y6–7.
a
School of Chemistry, University of Edinburgh, Joseph Black Building, King’s Buildings, West Mains Road, Edinburgh, UK EH3 9JJ. E-mail: [email protected]; Tel: +44 (0)131 650 7533 b School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK B15 2TT. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc46356g
198 | Chem. Commun., 2014, 50, 198--200
Fig. 1 Schematics showing ECD fragmentation channels for the three L4PL4K constructs. (A) Nomenclature of a–x, b–y, c–z and w fragment ions; (B) free-termini peptide; (C) Ac–COOH peptide; (D) Ac–OMe peptide.
Addition of an N-terminal acetyl group results in loss of the capping group, suppresses y9, and eliminates all c fragments (c2–5) due to the lowered proton affinity of the capped N-terminus. The nature of the C-terminus also affects the observable pattern, with the acetylated-free acid construct giving rise to z2, b3, b4, a6 and a7 and the construct with a methyl ester C-terminal group yielding z5 and z7–9, which were not detected for the former peptide. van der Rest et al.7 recently analysed the SwedECD8 and SwedCAD9 databases and measured the fragments of a family of synthetic peptides and concluded that formation of w ions may, despite expectations, proceed independently of z ion formation. Two distinct pathways may be identified for b–y ion formation: (1) secondary fragmentation of [M H]+ ions after H loss following electron capture10 producing a vibrationally and configurationally excited [M + H]+ ion which fragments via the mobile proton pathway encountered in CID; or (2) b–y ions may arise from peptide ions protonated on backbone nitrogen atoms.11 For all L4PL4K constructs b and y fragment ions are more prevalent following CID (Fig. S2, ESI†) than following ECD. CID of all peptides studied here does not produce any of the w ions observed by ECD. Therefore, an alternative explanation is required for rationalising the origin of these fragments. Julian et al.12,13 have reported side-chain losses from leucine residues after conversion of the ‘‘hydrogen abundant’’ radical into a ‘‘hydrogen deficient’’ state, accompanied by radical migration in the peptide ion, also reporting the formation of (usually low-abundance)
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b and y fragments from ‘‘hydrogen deficient’’ radicals. Radical migration may occur via a H jumping to form more stable radical sites and has been associated with the occurrence of H loss.12 The ECD spectra (see ESI,† Fig. S1) do display losses of both 43 Da and 44 Da from leucine residues, which may result from the ‘‘H deficient’’ and the ‘‘H abundant’’ pathway respectively. In any case, these fragmentation routes do occur via multi-step pathways that involve hydrogen atom mobility in the peptide ion. The potential of radical migration to probe non-covalent structures in polypeptides has already been noted.14 The following question arises: why are these fragments observed, and how can they be interpreted in terms of peptide conformation? We have performed IM-MS and molecular dynamics simulations to investigate the conformation(s) adopted by this hydrophobic peptide in the gas phase. Experimental and theoretical collision cross-sections (CCS) are depicted in Table 1, and Fig. 2 shows the lowest energy candidate geometries. Ion mobility analysis of polypeptides commonly reveals an increase in collision cross-section with increasing charge due to coulombic effects. This can be indicative of partially denatured forms present in solution or upon desolvation. In contrast, the collision cross-section of singly charged monomeric L4PL4K is never found to be smaller than that of the doubly protonated peptide ion for all constructs studied experimentally (Table 1). In fact the reverse is true: for the free-termini L4PL4K, the collision crosssection of the [M + 2H]2+ ion is 9.6% smaller than that of the [M + H]1+ ion; the relative difference is 7.4% for the Ac/COOH Table 1 Experimentally determined and theoretical (Boltzmann-weighted average of 500 potential energy minima) collision cross-sections for the L4PL4K constructs
Collision cross-sectiona (Å2) Construct
Exp. 1+
Theory 1+
Exp. 2+
Theory 2+
Free termini
297.9 1.89
297.1
271.7 0.63
Ac–COOH Ac–OMe
306.8 8.98 308.2 3.14
286.7 309.2
285.4 1.97 295.5 5.41
281.0b 294.4c 300.4c 306.6c
a Experimental errors are equal to 2 standard deviations based on three repeats. b Protonated at the N-terminal amine and at the K10 amine. c Protonated at the N-terminal amine and on the P5 carboxyl.
Fig. 2 Lowest energy minima for the free-termini L4PL4K peptide generated by simulated annealing. (A) [M + H]1+, (B) [M + H]2+ protonated at K10 and at the N-terminus and (C) [M + H]2+ protonated at K10 and P5. Leucine side-chains as well as protons of P5 and K10 side-chains are not shown for clarity.
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capped peptide and 4.3% for the Ac/OMe capped peptide (for the latter the difference is similar to the experimental error). Crosssections of the [M + H]1+ ion are very similar for the two capped constructs with the peptide with unprotected termini being somewhat smaller. Differences between [M + 2H]2+ ions are more noticeable. These results imply that [M + H]+ species have a particular structural preference that is altered by the addition of a single proton, the effect becoming pronounced for free-termini L4PL4K. Charge location strongly influences helix formation in vacuo. Hydrogen bonds between a charge and backbone hydroxyl groups at the C-terminus of the peptide will template helix formation. Conversely, protonation near the N-terminus – a likely scenario for L4PL4K [M + 2H]2+ ions with a free N-terminus – destabilises helical configurations.15 Molecular modelling was carried out to explain this phenomenon. As expected, [M + H]+ ions containing only a protonated side-chain amine at K10 (the peptide termini being either neutral or capped) are predominantly helical; however, positioning a charge at the N-terminus (as in the case of the [M + H]+ zwitterionic peptide or the [M + 2H]2+ ion containing a charge at the N-terminal amine) reduces the stability of the helix significantly. An inverse correlation between the helical content of the peptide (comprising of residues with either the 310-, a- or p-helical character as calculated by the DSSP model16) and potential energy can be seen for all constructs not containing a charged N-terminus (ESI,† Fig. S3 and S4). In these structures the helix is stabilised by hydrogen bonding interactions between the K10 side-chain and the backbone carbonyls of L7 and L8 (Fig. 2A). In peptides containing free termini the charge in the [M + 2H]2+ ion can be located either at the N-terminus or at the backbone amine and carbonyl groups, giving rise to multiple ‘‘charge isomers’’, of which only two are simulated here; (1) a protonated N-terminal amine [Fig. 2B and Table 1, footnote b] and (2) a protonated backbone at P5 [Fig. 2C and Table 1, footnote c] (in this case the proton was attached at the backbone carbonyl group yielding an iminium enol structure). P5 was chosen (the proton was added at the backbone carbonyl) since it appears to be more basic than leucine in published gas-phase basicity tables.17 Interestingly, the low-energy geometries of all [M + 2H]2+ ions protonated at K10 and P5 also tend to be helical. Inspection of the structures showed that the hydroxyl at the protonated backbone of P5 forms a hydrogen bond with the carbonyl of L9 and occasionally with that of K10, allowing a helical configuration to be retained. However, the [M + 2H]2+ construct protonated at the N-terminus adopts a collapsed structure in which the two charges are stabilised by seeking interactions with backbone carbonyls. The encasement of the N-terminal amine by the peptide in this construct prevents the formation of helical structures. Positioning a proline residue in the middle of a helix is expected to destabilise it due to the fact that tertiary amine of proline cannot participate in hydrogen bonding essential for helix formation. However in the range of L4PL4K variants studied here this loss is partially compensated: the carbonyl of L1 (which would form a hydrogen bond with X5 amide in an a-helical configuration) can interact instead with the amide NH of L4, in a 310-like arrangement enabling a proline containing a and 310 helices to be stable.
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In terms of the collision cross-section, the predicted value for simulated [M + H]+ agrees very well with the one determined experimentally (Table 1), with the possible exception of Ac–COOH. Thus despite the proline residue, the experimental data are consistent with helix formation in the gas phase. For the [M + 2H]2+ ions, greater discrepancies between experimental and simulated crosssections are observed. These differences can be attributed to more conformations and possibly different protonation sites are present experimentally than sampled here. However, the Ac–COOH peptide is not entirely collapsed (its cross-section is similar to that of the free-termini peptide protonated at P5 and K10), a feature that may be attributed to partial retention of the helix. Molecular dynamics simulations were also performed for these ions: the secondary structural preferences indicated by simulated annealing persisted during MD runs; the average helical content per residue for each construct, including the Ac–NH2 capped L9K 1+ peptide, which forms a stable helix, is shown in Fig. S5 (ESI†). Clearly, the proline residue, in particular if protonated, will destabilise helical configurations relative to L9K; but it should be noted that [M + 2H]2+ simulated with a charge at P5 and K10 is also predominantly helical. In these constructs destabilisation of the helix occurs over the first three residues; however the proline residue itself is incorporated into the helical stretch. As expected, charge location plays a pivotal role in determining the conformation of these short peptides. All constructs with identical charge localisation participate in the same non-covalent interactions, as seen in the a-carbon contact maps (ESI,† Fig. S6). The conformational preferences of the [M + H]+ ion, together with the prediction that at least some population of [M + 2H]2+ ions favour helicity, suggest that capture of an electron by [M + 2H]2+ ions is accompanied by refolding the charge-reduced species to form a helix with associated dipole moment. Recent reports have shown that helix formation in solution occurs on the nanosecond time scale18 and it is feasible that in the FT-ICR cell refolding occurs within the microsecond time scale of relaxation from high-n Rydberg states of the captured electron19 restructuring [M + 2H] + to a helical form akin to that found by ion mobility for the [M + H]+ ion. It has been suggested that internal dipoles can guide the trajectory of the incoming electron and stabilise the amide p* or S–S s* orbitals,20,21 and the helical propensity of the L4PL4K ion would certainly favour such a mechanism. Turecek and co-workers observed abnormal dissociation in phosphopeptides, specifically abundant loss of H atoms.21 The observed fragmentation of L4PL4K may also be attributed to a similar effect, i.e. formation of [M + H]+ ions following H loss and subsequent fragmentation to b–y ions, presumably via the mobile proton pathway. The observed correlation between b ion formation and helicity in L4PL4K is an intriguing feature and further studies may show if this property generalises in other helix-forming sequences as well. In order to probe the possibility of L4PL4K providing unusual ECD pathways by entropically stabilising the radical cation due to the size of Leu residues, the ECD spectrum of A7K [M + 2H]2+ was also measured (ESI,† Fig. S7). A7K led to the formation of b4–7, which is consistent with the features observed for L4PL4K. Notably, the previously measured ECD spectra of KL4PL4 and RL4PL4, whose singly charged ions are
200 | Chem. Commun., 2014, 50, 198--200
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not expected to be helical due to the side chain with the highest gas-phase basicity being located at the N-terminal end, do not give rise to b ions as well.22 A systematic survey of peptide sequences may reveal if this characteristic can indeed be generalised; in the cases that have been studied it appears that the conformational characteristics of the reduced ion can affect the ECD pathway. Peptide helices can be stabilised in the absence of solvent even when containing a helix-breaking residue such as proline in the middle of the sequence. Charge location also determines which configurations are stable in vacuo, which in turn impact other processes, such as fragmentation pathways, dependent on the network of intramolecular interactions that exist in the isolated ions. Here it appears that the helix forming propensity of the L4PL4K ions is responsible for the lack of c and z fragments and the increase in more CID-like fragments (b and y ions). This interesting observation may apply to other helical stretches of polypeptides and proteins and if so has implications on the use of ECD for top-down sequencing of intact proteins. ECD fragmentation is sensitive to secondary structure of both the precursor ions and also any long-lived intermediates. If atypical ECD spectra result for other peptide ions with helical intermediates, as for L4PL4K, then this information may be exploited to elucidate the secondary structure.
Notes and references 1 K. Breuker and F. W. McLafferty, Angew. Chem., Int. Ed., 2005, 44, 4911–4914. 2 K. Breuker, H. B. Oh, C. Lin, B. K. Carpenter and F. W. McLafferty, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 14011–14016. 3 E. Jurneczko and P. E. Barran, Analyst, 2011, 136, 20–28. 4 R. A. Zubarev, N. L. Kelleher and F. W. McLafferty, J. Am. Chem. Soc., 1998, 120, 3265–3266. 5 R. A. Zubarev, D. M. Horn, E. K. Fridriksson, N. L. Kelleher, N. A. Kruger, M. A. Lewis, B. K. Carpenter and F. W. McLafferty, Anal. Chem., 2000, 72, 563–573. 6 F. Kjeldsen, K. F. Haselmann, B. A. Budnik, F. Jensen and R. A. Zubarev, Chem. Phys. Lett., 2002, 356, 201–206. 7 G. van der Rest, R. J. Hui, G. Frison and J. Chamot-Rooke, J. Am. Soc. Mass Spectrom., 2011, 22, 1631–1644. 8 M. M. Savitski, M. L. Nielsen and R. A. Zubarev, Anal. Chem., 2007, 79, 2296–2302. 9 M. Falth, M. M. Savitski, M. L. Nielsen, F. Kjeldsen, P. E. Andren and R. A. Zubarev, J. Proteome Res., 2007, 6, 4063–4067. 10 I. Swierszcz, P. Skurski and J. Simons, J. Phys. Chem. A, 2012, 116, 1828–1837. 11 V. Bakken, T. Helgaker and E. Uggerud, Eur. J. Mass Spectrom., 2004, 10, 625–638. 12 B. N. Moore, T. Ly and R. R. Julian, J. Am. Chem. Soc., 2011, 133, 6997–7006. 13 Q. Y. Sun, H. Nelson, T. Ly, B. M. Stoltz and R. R. Julian, J. Proteome Res., 2009, 8, 958–966. 14 T. Ly and R. R. Julian, J. Am. Chem. Soc., 2010, 132, 8602–8609. 15 R. R. Hudgins, M. A. Ratner and M. F. Jarrold, J. Am. Chem. Soc., 1998, 120, 12974–12975. 16 W. Kabsch and C. Sander, Biopolymers, 1983, 22, 2577–2637. 17 A. G. Harrison, Mass Spectrom. Rev., 1997, 16, 201–217. 18 D. De Sanco and R. B. Best, J. Am. Chem. Soc., 2011, 133, 6809–6816. 19 J. Simons, Chem. Phys. Lett., 2010, 484, 81–95. 20 I. Swierszcz, P. Skurski and J. Simons, J. Phys. Chem. A, 2012, 116, 1828–1837. 21 C. L. Moss, T. W. Chung, J. A. Wyer, S. Bronsted Nielsen, P. Hvelplund and F. Turecek, J. Am. Soc. Mass Spectrom., 2011, 22, 731–751. 22 H. J. Cooper, J. Am. Soc. Mass Spectrom., 2005, 16, 1932–1940.
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Cite this: Chem. Commun., 2014, 50, 198 Received 19th August 2013, Accepted 15th October 2013
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Unusual ECD fragmentation attributed to gas-phase helix formation in a conformationally dynamic peptide† Jason M. D. Kalapothakis,a Yana Berezovskaya,a Cleidiane G. Zampronio,b Peter A. Faull,a Perdita E. Barran*a and Helen J. Cooper*b
DOI: 10.1039/c3cc46356g www.rsc.org/chemcomm
The helix-forming character of a model decapeptide, L4PL4K, is determined in the absence of solvent using ion mobility mass spectrometry, electron capture dissociation and molecular mechanics simulations. Unusual ECD fragmentation patterns dominated by b ions are attributed to helix formation upon electron capture and as a signature of conformational dynamics.
In recent years there has been an increase in the use of gas phase methods to determine higher order structures of biological molecular systems.1–3 Much of this work requires biological macromolecules to maintain their structure over short (> ms) experimental time scales; however this premise suffers in those cases where a system possesses fast conformational dynamics. In this work a simple model decapeptide, L4PL4K, is examined using ion mobility mass spectrometry (IM-MS), electron capture dissociation (ECD) and molecular mechanics simulations in order to delineate the effects of helix forming propensity on fragmentation behaviour. Following ECD (see ESI† for experimental details), the most notable feature of L4PL4K ECD fragmentation is the prominence of CID-like fragments, w fragments and H loss. Typically ECD yields c0 and z type fragments with cleavage along the N–Ca bond4,5 (Fig. 1A); however, L4PL4K constructs exhibit ECD spectra in which c and z fragment ions are relatively sparse, whereas b, y ion pairs as well as w ions (thought to result from higher-energy dissociation processes6) are most persistent (Fig. 1B–D and Fig. S1, ESI†). Side chain losses are prominent across all three constructs, with w6–10 and w5–4 being present in all ECD spectra of [M + 2H]2+ ions. These fragments are detected even in cases where the corresponding z ions are absent, as they are for the free termini and acetyl-free acid constructs w5, w7 and w8. Other ubiquitous fragments across all three constructs are z4, b2–5, b9 and y6–7.
a
School of Chemistry, University of Edinburgh, Joseph Black Building, King’s Buildings, West Mains Road, Edinburgh, UK EH3 9JJ. E-mail: [email protected]; Tel: +44 (0)131 650 7533 b School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK B15 2TT. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc46356g
198 | Chem. Commun., 2014, 50, 198--200
Fig. 1 Schematics showing ECD fragmentation channels for the three L4PL4K constructs. (A) Nomenclature of a–x, b–y, c–z and w fragment ions; (B) free-termini peptide; (C) Ac–COOH peptide; (D) Ac–OMe peptide.
Addition of an N-terminal acetyl group results in loss of the capping group, suppresses y9, and eliminates all c fragments (c2–5) due to the lowered proton affinity of the capped N-terminus. The nature of the C-terminus also affects the observable pattern, with the acetylated-free acid construct giving rise to z2, b3, b4, a6 and a7 and the construct with a methyl ester C-terminal group yielding z5 and z7–9, which were not detected for the former peptide. van der Rest et al.7 recently analysed the SwedECD8 and SwedCAD9 databases and measured the fragments of a family of synthetic peptides and concluded that formation of w ions may, despite expectations, proceed independently of z ion formation. Two distinct pathways may be identified for b–y ion formation: (1) secondary fragmentation of [M H]+ ions after H loss following electron capture10 producing a vibrationally and configurationally excited [M + H]+ ion which fragments via the mobile proton pathway encountered in CID; or (2) b–y ions may arise from peptide ions protonated on backbone nitrogen atoms.11 For all L4PL4K constructs b and y fragment ions are more prevalent following CID (Fig. S2, ESI†) than following ECD. CID of all peptides studied here does not produce any of the w ions observed by ECD. Therefore, an alternative explanation is required for rationalising the origin of these fragments. Julian et al.12,13 have reported side-chain losses from leucine residues after conversion of the ‘‘hydrogen abundant’’ radical into a ‘‘hydrogen deficient’’ state, accompanied by radical migration in the peptide ion, also reporting the formation of (usually low-abundance)
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b and y fragments from ‘‘hydrogen deficient’’ radicals. Radical migration may occur via a H jumping to form more stable radical sites and has been associated with the occurrence of H loss.12 The ECD spectra (see ESI,† Fig. S1) do display losses of both 43 Da and 44 Da from leucine residues, which may result from the ‘‘H deficient’’ and the ‘‘H abundant’’ pathway respectively. In any case, these fragmentation routes do occur via multi-step pathways that involve hydrogen atom mobility in the peptide ion. The potential of radical migration to probe non-covalent structures in polypeptides has already been noted.14 The following question arises: why are these fragments observed, and how can they be interpreted in terms of peptide conformation? We have performed IM-MS and molecular dynamics simulations to investigate the conformation(s) adopted by this hydrophobic peptide in the gas phase. Experimental and theoretical collision cross-sections (CCS) are depicted in Table 1, and Fig. 2 shows the lowest energy candidate geometries. Ion mobility analysis of polypeptides commonly reveals an increase in collision cross-section with increasing charge due to coulombic effects. This can be indicative of partially denatured forms present in solution or upon desolvation. In contrast, the collision cross-section of singly charged monomeric L4PL4K is never found to be smaller than that of the doubly protonated peptide ion for all constructs studied experimentally (Table 1). In fact the reverse is true: for the free-termini L4PL4K, the collision crosssection of the [M + 2H]2+ ion is 9.6% smaller than that of the [M + H]1+ ion; the relative difference is 7.4% for the Ac/COOH Table 1 Experimentally determined and theoretical (Boltzmann-weighted average of 500 potential energy minima) collision cross-sections for the L4PL4K constructs
Collision cross-sectiona (Å2) Construct
Exp. 1+
Theory 1+
Exp. 2+
Theory 2+
Free termini
297.9 1.89
297.1
271.7 0.63
Ac–COOH Ac–OMe
306.8 8.98 308.2 3.14
286.7 309.2
285.4 1.97 295.5 5.41
281.0b 294.4c 300.4c 306.6c
a Experimental errors are equal to 2 standard deviations based on three repeats. b Protonated at the N-terminal amine and at the K10 amine. c Protonated at the N-terminal amine and on the P5 carboxyl.
Fig. 2 Lowest energy minima for the free-termini L4PL4K peptide generated by simulated annealing. (A) [M + H]1+, (B) [M + H]2+ protonated at K10 and at the N-terminus and (C) [M + H]2+ protonated at K10 and P5. Leucine side-chains as well as protons of P5 and K10 side-chains are not shown for clarity.
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capped peptide and 4.3% for the Ac/OMe capped peptide (for the latter the difference is similar to the experimental error). Crosssections of the [M + H]1+ ion are very similar for the two capped constructs with the peptide with unprotected termini being somewhat smaller. Differences between [M + 2H]2+ ions are more noticeable. These results imply that [M + H]+ species have a particular structural preference that is altered by the addition of a single proton, the effect becoming pronounced for free-termini L4PL4K. Charge location strongly influences helix formation in vacuo. Hydrogen bonds between a charge and backbone hydroxyl groups at the C-terminus of the peptide will template helix formation. Conversely, protonation near the N-terminus – a likely scenario for L4PL4K [M + 2H]2+ ions with a free N-terminus – destabilises helical configurations.15 Molecular modelling was carried out to explain this phenomenon. As expected, [M + H]+ ions containing only a protonated side-chain amine at K10 (the peptide termini being either neutral or capped) are predominantly helical; however, positioning a charge at the N-terminus (as in the case of the [M + H]+ zwitterionic peptide or the [M + 2H]2+ ion containing a charge at the N-terminal amine) reduces the stability of the helix significantly. An inverse correlation between the helical content of the peptide (comprising of residues with either the 310-, a- or p-helical character as calculated by the DSSP model16) and potential energy can be seen for all constructs not containing a charged N-terminus (ESI,† Fig. S3 and S4). In these structures the helix is stabilised by hydrogen bonding interactions between the K10 side-chain and the backbone carbonyls of L7 and L8 (Fig. 2A). In peptides containing free termini the charge in the [M + 2H]2+ ion can be located either at the N-terminus or at the backbone amine and carbonyl groups, giving rise to multiple ‘‘charge isomers’’, of which only two are simulated here; (1) a protonated N-terminal amine [Fig. 2B and Table 1, footnote b] and (2) a protonated backbone at P5 [Fig. 2C and Table 1, footnote c] (in this case the proton was attached at the backbone carbonyl group yielding an iminium enol structure). P5 was chosen (the proton was added at the backbone carbonyl) since it appears to be more basic than leucine in published gas-phase basicity tables.17 Interestingly, the low-energy geometries of all [M + 2H]2+ ions protonated at K10 and P5 also tend to be helical. Inspection of the structures showed that the hydroxyl at the protonated backbone of P5 forms a hydrogen bond with the carbonyl of L9 and occasionally with that of K10, allowing a helical configuration to be retained. However, the [M + 2H]2+ construct protonated at the N-terminus adopts a collapsed structure in which the two charges are stabilised by seeking interactions with backbone carbonyls. The encasement of the N-terminal amine by the peptide in this construct prevents the formation of helical structures. Positioning a proline residue in the middle of a helix is expected to destabilise it due to the fact that tertiary amine of proline cannot participate in hydrogen bonding essential for helix formation. However in the range of L4PL4K variants studied here this loss is partially compensated: the carbonyl of L1 (which would form a hydrogen bond with X5 amide in an a-helical configuration) can interact instead with the amide NH of L4, in a 310-like arrangement enabling a proline containing a and 310 helices to be stable.
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In terms of the collision cross-section, the predicted value for simulated [M + H]+ agrees very well with the one determined experimentally (Table 1), with the possible exception of Ac–COOH. Thus despite the proline residue, the experimental data are consistent with helix formation in the gas phase. For the [M + 2H]2+ ions, greater discrepancies between experimental and simulated crosssections are observed. These differences can be attributed to more conformations and possibly different protonation sites are present experimentally than sampled here. However, the Ac–COOH peptide is not entirely collapsed (its cross-section is similar to that of the free-termini peptide protonated at P5 and K10), a feature that may be attributed to partial retention of the helix. Molecular dynamics simulations were also performed for these ions: the secondary structural preferences indicated by simulated annealing persisted during MD runs; the average helical content per residue for each construct, including the Ac–NH2 capped L9K 1+ peptide, which forms a stable helix, is shown in Fig. S5 (ESI†). Clearly, the proline residue, in particular if protonated, will destabilise helical configurations relative to L9K; but it should be noted that [M + 2H]2+ simulated with a charge at P5 and K10 is also predominantly helical. In these constructs destabilisation of the helix occurs over the first three residues; however the proline residue itself is incorporated into the helical stretch. As expected, charge location plays a pivotal role in determining the conformation of these short peptides. All constructs with identical charge localisation participate in the same non-covalent interactions, as seen in the a-carbon contact maps (ESI,† Fig. S6). The conformational preferences of the [M + H]+ ion, together with the prediction that at least some population of [M + 2H]2+ ions favour helicity, suggest that capture of an electron by [M + 2H]2+ ions is accompanied by refolding the charge-reduced species to form a helix with associated dipole moment. Recent reports have shown that helix formation in solution occurs on the nanosecond time scale18 and it is feasible that in the FT-ICR cell refolding occurs within the microsecond time scale of relaxation from high-n Rydberg states of the captured electron19 restructuring [M + 2H] + to a helical form akin to that found by ion mobility for the [M + H]+ ion. It has been suggested that internal dipoles can guide the trajectory of the incoming electron and stabilise the amide p* or S–S s* orbitals,20,21 and the helical propensity of the L4PL4K ion would certainly favour such a mechanism. Turecek and co-workers observed abnormal dissociation in phosphopeptides, specifically abundant loss of H atoms.21 The observed fragmentation of L4PL4K may also be attributed to a similar effect, i.e. formation of [M + H]+ ions following H loss and subsequent fragmentation to b–y ions, presumably via the mobile proton pathway. The observed correlation between b ion formation and helicity in L4PL4K is an intriguing feature and further studies may show if this property generalises in other helix-forming sequences as well. In order to probe the possibility of L4PL4K providing unusual ECD pathways by entropically stabilising the radical cation due to the size of Leu residues, the ECD spectrum of A7K [M + 2H]2+ was also measured (ESI,† Fig. S7). A7K led to the formation of b4–7, which is consistent with the features observed for L4PL4K. Notably, the previously measured ECD spectra of KL4PL4 and RL4PL4, whose singly charged ions are
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not expected to be helical due to the side chain with the highest gas-phase basicity being located at the N-terminal end, do not give rise to b ions as well.22 A systematic survey of peptide sequences may reveal if this characteristic can indeed be generalised; in the cases that have been studied it appears that the conformational characteristics of the reduced ion can affect the ECD pathway. Peptide helices can be stabilised in the absence of solvent even when containing a helix-breaking residue such as proline in the middle of the sequence. Charge location also determines which configurations are stable in vacuo, which in turn impact other processes, such as fragmentation pathways, dependent on the network of intramolecular interactions that exist in the isolated ions. Here it appears that the helix forming propensity of the L4PL4K ions is responsible for the lack of c and z fragments and the increase in more CID-like fragments (b and y ions). This interesting observation may apply to other helical stretches of polypeptides and proteins and if so has implications on the use of ECD for top-down sequencing of intact proteins. ECD fragmentation is sensitive to secondary structure of both the precursor ions and also any long-lived intermediates. If atypical ECD spectra result for other peptide ions with helical intermediates, as for L4PL4K, then this information may be exploited to elucidate the secondary structure.
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