Creese and Cooper
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N-terminus and the observed fragmentation suggests that both situations occur, even for the
unmodified peptide. For those precursors, which contain deprotonated phospho groups, it is
likely salt bridges with protonated amino acid side chains exist. However, for most N-Cα
cleavages within the peptides, the resulting complementary fragments will be positively
charged and will separate as a result of Coulombic repulsion.
Figure 5 shows the ECD mass spectra obtained from doubly-charged ions of the β-casein
peptide (FQ(p)SEEQQQTEDELQDK). Figure 5a and b show the ECD mass spectra obtained
from the dephosphorylated peptide at ECD cathode potentials of -3.34 V (standard) and -9.34
V (i.e., the cathode potential at which greatest sequence coverage was observed). Figure 5c
and d show the equivalent ECD mass spectra obtained for the phosphopeptide. ECD of the
unmodified peptide at standard cathode potential (Figure 5a) resulted in cleavage of seven out
of 15 N-Ca bonds. For the phosphorylated peptide at standard ECD cathode potential (Figure
5c), cleavage of five out of the 15 N-Ca bonds was observed and the site of modification
identified. As with the synthetic peptides, the increase in cathode potential results in greater
peptide coverage. At a cathode potential of -9.34 V, complete sequence coverage was obtained
for the unmodified peptide. At a cathode potential of -12.84 V, 13 of the 15 N-Ca bonds were
cleaved in the phosphopeptide.
Unlike the phosphopeptides described above, these peptides have a single basic residue. The
sequence coverage for the phosphorylated peptide is less than for the unmodified version but
the difference cannot be accounted for by deprotonation of the phospho-group alone. These
peptides are highly acidic. For the unmodified peptide, the fragmentation pattern at standard
ECD energy (Figure 5a) suggests that Gln6 and Gln14 are protonated [51], Glu10 and Glu12
are deprotonated, and that salt bridges between the positively and negatively charged groups
prevent detection of the ECD fragments. The fragmentation pattern for the phosphorylated
peptide suggests that additional noncovalent interactions are present, e.g., that Glu4 is
deprotonated and bound via a salt bridge to a glutamine. It is postulated that the fragmentation
pattern reflects a change in conformation of the peptide as a result of phosphorylation, rather
than the direct involvement of the phospho-group in any noncovalent bonding. For both
peptides increasing the ECD electron energy results in a marked increase in backbone
fragments, particularly z ions. Higher electron energy is required by the phosphopeptide to
achieve maximum sequence coverage. The results suggest that deposition of additional energy
cleaves any noncovalent interactions, with associated hydrogen rearrangement, enabling
detection of the backbone fragments.
Similar results were obtained following ECD of doubly-charged ions of the a-S1-casein
phosphopeptide (YKVPQLEIVPNpSAEER) and its unmodified counterpart (see Figure 6).
The mass spectrum of the unmodified peptide at the standard ECD cathode potential (Figure
6a) reveals only four N-Ca fragments (c 15, c 14, and z 14∙, z 12∙). The fragmentation pattern
suggests that there is a noncovalent interaction between deprotonated Glu14 and protonated
Gln5 [51]. There were only three fragments (c 15, z 15∙, and z 14∙) observed following ECD of
the phosphorylated peptide at standard cathode potential (Figure 6c). The phosphorylation site
cannot be localized. The lack of fragments appears to be the result of a salt bridge between
protonated Pro4 [51] and a deprotonated glutamic acid residue, in this case Glu15. (Note that
we do not expect cleavage N-terminal to proline). As for the previous case, apparently the
effect of phosphorylation on conformation is key rather than the involvement of the phospho-
group in noncovalent bonding. The greatest sequence coverage of the unmodified peptide
(Figure 6b) was obtained with an ECD cathode potential of -11.84 V; 12 out of 15 N-Ca bonds
were cleaved and the y7 and y13 fragments were observed. The greatest coverage for the
phosphorylated peptide (Figure 6d) was observed at a cathode potential of -12.34 V. Eleven
out of fifteen N-Ca bonds were cleaved and the y7 and y13 fragments were produced. The site
Published as: J Am Soc Mass Spectrom. 2008 September ; 19(9): 1263-1274.