Ion-molecule reactions reveal facile radical migration in peptides

Article abstract of DOI:10.1039/b907833a

Ion-molecule reactions between molecular oxygen and peptide radicals in the gas phase demonstrate that radical migration occurs easily within large biomolecules without addition of collisional activation energy.

Full text of DOI:10.1039/b907833a

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Ion–molecule reactions reveal facile radical migration in peptidesw
Benjamin N. Moore,b Stephen J. Blanksby*a and Ryan R. Julian*b
Received (in Cambridge, UK) 20th April 2009, Accepted 30th June 2009
First published as an Advance Article on the web 13th July 2009
DOI: 10.1039/b907833a
Ion–molecule reactions between molecular oxygen and peptide
radicals in the gas phase demonstrate that radical migration
occurs easily within large biomolecules without addition of
collisional activation energy.
differentiating isomeric radical ions.20,21 Dioxygen has been
shown to be a selective reagent, reacting with distonic radical
ions (i.e., where the charge and unpaired electron are formally
localised on separate atoms within the molecule),22–24 to form
[M + O2] adduct ions while remaining unreactive toward
conventional radical ions.25–28 Furthermore, dioxygen is often
present as a background gas inside commercial mass spectro-
meters thus obviating the need for instrument modification to
observe these ion–molecule reactions. In a recent example,
McLuckey and co-workers found that peptide radical cations
resulting from electron transfer dissociation of multiply
charged polypeptides form [M + O2] adduct ions upon
storage in an ion-trap mass spectrometer. This allows for the
facile distinction between z-ions and their complementary
even-electron c-ion counterparts.29
With the continued development of mass spectrometric
methods employing radical chemistry to fragment peptides
and proteins in the gas phase, a detailed understanding of the
behaviour of radicals in gaseous peptide ions is vitally
important. Numerous methods (including electron initiated
fragmentation,1,2 direct bond homolysis by photolysis3 or
collisional activation,4–10 and photoionisation11) can be used
to generate peptide radicals. Following radical generation, the
subsequent fragmentation chemistry of the peptide is typically
dominated by radical-directed processes, making the location
of the radical crucial as the initiation point for fragmentation.
It is of paramount importance, therefore, to determine
whether radicals can migrate within the framework of a
peptide or whether barriers exist to prevent such migration.
Recent work, based on the comparison of collisional
activation mass spectra of isomeric triglycine radical
peptides and accompanying electronic structure calculations,
concluded that radical migration is restricted by substantial
activation barriers.12 In contrast, other computational studies
have estimated significantly smaller barriers.13–15 In addition,
a body of indirect experimental evidence also suggests that
radical migration occurs in peptides prior to fragmentation.16–18
However, experiments requiring collisional activation or other
fragmentation methods are an imperfect probe of the structure
of the nascent radical ions given that the possibility of radical
migration (over substantial activation barriers), immediately
prior to fragmentation, cannot be excluded. Herein, we
demonstrate that reactive radicals generated within peptide
cations can migrate immediately after their formation and
without additional activation energy. This has been achieved
through the novel combination of photodissociation for the
regioselective generation of peptide radical cations and the use
of ion–molecule reactions to probe the final location(s) of
the radical.
We have previously demonstrated that peptide radical
cations can be generated by the gas phase photodissociation
of the C–I bond within iodinated tyrosine residues (see ESIw).3
Thus formed, these radicals yield site-specific fragmentation
upon subsequent collisional activation. This method provides
a known starting point for the radical at the 3-position of the
tyrosine side-chain. In this study, the archetypal tyrosinyl
radical cation, [Tyrꢀ + H]+ (m/z 181), was generated by laser
irradiation (266 nm) of protonated 3-iodotyrosine in a
specially modified linear ion-trap mass spectrometer. The
CID spectrum of [Tyrꢀ + H]+ (Fig. 1a) shows neutral losses
of 17 and 46 Da that have previously been assigned for
the even-electron [Tyr + H]+ cation to losses of NH3 and
[CO + H2O], respectively.30,31 The absence of specific radical-
driven dissociation products in Fig. 1a is consistent with
localisation of the unpaired electron at the 3-position on the
phenyl moiety: with no evidence for radical migration even
upon application of activation energy. Fig. 1b shows the mass
spectrum obtained from trapping [Tyrꢀ + H]+ in the presence
of adventitious O2 for 3 s. The absence of a significant
+
[M + O2]ꢀ adduct ion at m/z 213 contrasts with previous
studies of distonic radical cations (see earlier), as well as, the
analogous reaction of O2 with the phenylalaninyl radical
cation, [Pheꢀ + H]+, formed via photolysis of protonated
4-iodophenylalanine (Fig. 1c). The presence of an abundant
[M + 15] ion in Fig. 1b suggests distinctive reactivity for
[Tyrꢀ + H]+ that can be rationalised in terms of addition of
O2 coupled with the elimination of HOꢀ facilitated by the
adjacent hydroxyl group (Scheme 1).
Gas phase ion–molecule reactions have been used
extensively in mass spectrometry as probes of molecular
structure,19 and have proven particularly powerful in
a School of Chemistry, University of Wollongong, Wollongong, NSW,
Australia. E-mail: blanksby@uow.edu.au; Fax: +61 2 4221 5484;
Tel: +61 2 4221 5484
This mechanism is supported by (i) the observation of the
expected DOꢀ loss in the spectrum of the [D4 À Tyrꢀ + D]+
isotopologue and (ii) the retention of additional oxygen with
the phenol moiety in the m/z 123 fragment ion formed by
subsequent dissociation of the energised m/z 196 product ion
(see ESIw). Interestingly, the putative structure of the m/z 196
b Department of Chemistry, University of California Riverside,
Riverside, CA, USA. E-mail: ryan.julian@ucr.edu;
Fax: +1 951 827 2435; Tel: +1 951 827 3958
w Electronic supplementary information (ESI) available: Experimental
section, kinetics data, deuteration data. See DOI: 10.1039/b907833a
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Fig. 2 (a) The spectrum resulting from the gas phase reaction of
[RGYALG + H]ꢀ+, formed via photodissociation (266 nm) of
[RGY(I)ALG + H]+, with O2. The analogous spectra for the radical
Fig.
1
(a) Collision-induced dissociation mass spectrum of
[Tyrꢀ + H]+. (b) The spectrum resulting from the gas phase
reaction of [Tyrꢀ + H]+, formed via photodissociation (266 nm) of
[Tyr(I) + H]+, with O2. (c) The spectrum resulting from the gas phase
reaction of [Pheꢀ + H]+, formed via photodissociation (266 nm) of
[Phe(I) + H]+, with O2.
+
6+
cations (b) [DRVYIHPF + H]ꢀ and (c) [ubiquitin + 6H]ꢀ
.
Significantly, in this spectrum the distinctive [M + O2 À HOꢀ]+
is of low abundance, suggesting few, if any, of these ions retain
the radical in the 3-position on tyrosine. Furthermore, the
+
observation of the [M + O2]ꢀ adduct ion at m/z 667
indicates the presence of an alternative reactive isomer in the
radical ion population. Collision-induced dissociation of the
[RGYALG + H + O2]ꢀ+ at m/z 667 yields m/z 650, indicative
of HOꢀ loss, while deuterium labelling of this peptide at the
a-carbon on leucine results in DOꢀ loss under the same
experimental conditions (see ESIw). This suggests that the
m/z 667 ion includes a peroxyl radical at leucine, resulting
from migration of the radical to this residue prior to the
reaction with O2 (cf. Scheme 2). Deuterium labelling on the
g-carbon of leucine confirms direct transfer of the radical from
tyrosine to leucine (ESIw). While these data indicate radical
migration to leucine can occur, the presence of other isomers
resulting from rearrangement of the nascent radical cation
cannot be excluded. Indeed, pseudo-first order kinetic analysis
Scheme 1 Proposed mechanism for the reaction of [Tyrꢀ + H]+ with
dioxygen.
product ion corresponds to protonated dopaquinone: a
reactive intermediate in the tyrosinase catalysed oxidation of
tyrosine.32,33 Taken together these data provide strong
evidence for the localisation of the unpaired electron in the
3-position. Furthermore, kinetic analysis of the reaction of m/z
181 with dioxygen yields strictly linear first-order kinetics with
a second order rate constant of 2.06 Â 10À11 moleculesÀ1 cm3 sÀ1
and a reaction efficiency of 3.6% (see ESIw). This behaviour is
indicative of a single, reactive radical cation at m/z 181 and we
conclude that photodissociation of protonated 3-iodotyrosine
at 266 nm yields a single phenyl-type radical structure that
undergoes a distinctive concerted reaction in the presence of
dioxygen as indicated in Scheme 1. The absence of exothermic
rearrangements to stabilised radicals (e.g., phenoxyl, a- or
b-radicals) suggests significant activation barriers exist for
hydrogen atom transfer within this isolated amino acid.
The hexapeptide radical cation [RGYꢀALG + H]+ was
synthesised via photodissociation of the 3-iodotyrosine
analogue. Fig. 2a shows the mass spectrum obtained when
this ion is trapped in the presence of O2 for a period of 3 s.
+
of the reaction of [RGYALG + H]ꢀ with dioxygen reveals
significant curvature suggesting the ion population consists of
at least two isomeric radical cations (see ESIw); (i) a fast
reacting radical with the unpaired electron likely localised on
the leucine side-chain and (ii) a slow or unreactive isomer.
Radical migration from the 3-positon on tyrosine to any of the
a-position(s) along the peptide backbone is predicted to be
exothermic by as much as 30 kcal mol .
À1 34 Such radicals may
Scheme 2 Proposed mechanism for the reaction of [RGYALꢀG + H]+
with dioxygen.
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+
account for the less reactive fraction of the [RGYALG + H]ꢀ
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ion population. Indeed, the authentic a-radicals (at least
initially), formed by collision-induced side-chain cleavage,
are substantially less reactive or unreactive with O2 (see ESIw).
We therefore conclude that following photodissociation of
the C–I bond on the iodinated tyrosine residue, the radical
does not remain localised at the 3-position but rather migrates
to one or more locations throughout the peptide. Similar
behaviour is observed for the reaction of the angiotensin
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+
radical cation [DRVYIHPF + H]ꢀ (Fig. 2b) and even
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for multiply charged ubiquitin (Fig. 2c). The presence of
[M + O2]+ adduct ions in these spectra is indicative of the
presence of radicals at sites remote from the initial location on
tyrosine.
Interestingly, the spectra shown in Fig. 2a–c also reveal
significant amounts of side-chain fragmentation similar to
pathways previously described for the CID of peptide
radicals,34 e.g., the loss of the isobutene (À56 Da) from
the leucine side-chain in Fig. 2a. Furthermore, these frag-
mentation pathways reveal a time-dependence (see ESIw)
and appear to be independent of the concentration of
dioxygen (the latter was established by comparing the
[RGYALG + O2]ꢀ and [RGYALG À 56]ꢀ+ ion abundances
+
while varying the availability of air in the electrospray source
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The results presented herein unambiguously demonstrate
that radical migration can occur in large peptides without the
addition of significant activation energy. The possibility for
facile radical migration must therefore be considered in
fragmentation experiments where radical peptides are
generated. If needed, ion–molecule reactions such as those
demonstrated above can provide a facile route to monitor
radical migration in peptides.
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S. J. B. acknowledges funding from the Australian Research
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National Science Foundation (CHE-0747481).
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