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Scheme 2. Peptide Formation of ac-AAAAK Pentapeptides
from the [ac-AAA+AK+H]+ Complex upon 157 nm Irradition
m/z = 147.0, 214.0, 218.1, 257.2, 285.2, 289.2, and 360.1
fragments can be assigned to the y1, b3, y2, a4, b4, y3, and y4 ions
of AAAAK.
This interpretation requires that it is possible to eliminate
water from either end of the complex (Scheme 1) and form
peptide bonds that link the smaller units together to produce a
longer amino acid chain. In this sense, the proton-bound com-
plex presents itself as a well-defined, long-lived intermediate
along the pathway to larger peptide formation. With this in mind,
it should be possible to control which sequence is formed by
simply blocking the end of one of the peptides. This prevents
water elimination from one head-to-tail region of the complex, as
shown in Scheme 2, where the amino terminus of the precursor
AAA peptide is acetylated, to produce ac-AAA.
Figure 2 shows the results of experiments that are analogous to
those described above; however, we first have acetylated the
amino terminus of the tripeptide AAA. By blocking this group,
photoexcitation of the noncovalent complex should produce
only the ac-AAAAK sequence. As discussed above, we first select
the proton-bound dimer precursor (Figure 2a) as the proposed
intermediate for [ac-AAAAK+H]+ formation. A CID analysis
confirms that this complex corresponds to the noncovalent
[ac-AAA+AK+H]+ complex (Figure 2b). Upon 157 nm irradia-
tion, a small peak corresponding to the [MÀ18+H]+ water-loss
product at m/z = 473.3 is detected (Figure 2c). Finally, Figure 2d
shows the CID spectrum of the m/z = 473.3 ion. This spectrum is
remarkably simple, and a nearly complete set of y and b ions
corresponding to only the ac-AAAAK sequence is observed.
Additionally, this spectrum is indistinguishable from that ob-
tained upon fragmentation of [ac-AAAAK+H]+ ions, where the
precursor ac-AAAAK peptide (here, used as a standard) was
produced by solid-phase synthesis. The AKAAA sequence has
clearly been suppressed by acetylating the sequence AAA,
providing exquisite reaction control. It is noteworthy that no
evidence for cross-linking involving the lysine residue butylamine
group is observed.
Figure 1. (a) Isolation spectrum of the [AAA+AK+H]+ complex ions,
and MS/MS spectra of [AAA+AK+H]+ obtained by (b) CID and
(c) 157 nm irradiation. Product ions from the [AK+H]+ and [AAA+H]+
are highlighted in red and blue, respectively. # indicates the loss of water.
(d) CID spectrum of the water-loss product (M#) in spectrum c. Peaks
are assigned according to the two possible peptide sequences, AAAAK
(red) and AKAAA (blue).
dissociate, primarily by the loss of the AAA neutral, producing the
large [AK+H]+ peak (m/z = 218.2). The AK neutral loss channel,
resulting in [AAA+H]+ (m/z = 232.2), is also observed, but the
ion signal is much smaller. This analysis is consistent with our
assignment of [AAA+AK+H]+ as a noncovalent complex.
When the accumulated [AAA+AK+H]+ complexes are irra-
diated with the F2 laser, we obtain the fragment ion spectrum
shown in Figure 1c. In addition to peaks observed from dissocia-
tion of the noncovalent complex, many new types of ions that
arise from breaking covalent bonds can be observed, similar to
those previously reported in 157 nm photofragmentation studies
of peptide monomer ions.25,26 One difference that stands out is
the observation of the [MÀ18+H]+ peak (M#) at m/z = 431.2.
This ion must arise from the loss of neutral water. While elimi-
nation of water or ammonia upon thermal activation of peptide
monomer ions is well known,27 there are no reports of this
phenomenon for noncovalent peptide complexes. These types of
species normally dissociate to form monomer units.
This phenomenon is observed for a range of amino acid chain
lengths and sequences, as shown in Table 1. Thus, we can direct
the formation of a range of well-known or exotic species for
study. For example, combination of the pentapeptide ac-RPPGF
and tetrapeptide SPFR can be used to produce the nonapeptide
ac-RPPGFSPFR (acetylated bradykinin). Alternatively, inclusion
of unnatural amino acids into the noncovalent intermediate com-
plex allows formation of species such the tripeptide ac-AAA
that incorporates a dodecylamine group at the C-terminal end
(Table 1). Finally, it is well known that, by incorporating more
To understand the origin of water elimination, we have colli-
sionally activated the [MÀ18+H]+ ion (M#) that appears from
photoexcitation(Figure1c). The resulting CID spectrum isshown
in Figure 1d. Interpretation of this spectrum is somewhat com-
plicated. Assuming that both [AKAAA+H]+ and [AAAAK+H]+
are formed, peaks at m/z = 200.0, 342.2, and 360.1 correspond
to the b2, b4, and y4 ions from the AKAAA sequence. The
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dx.doi.org/10.1021/ja205471n |J. Am. Chem. Soc. 2011, 133, 15834–15837