he Effect of Phosphorylation on the Electron Capture Dissociation of Peptide Ions

average fragment ion mass. In the case of the singly-phosphorylated peptide
(APLSFRGSLPK_pSYVK) (Figure 3b), the larger fragments, c 14, c ₁₃, c 12, z 12∙, z 11∙, and z 1₀∙
showed an overall decrease in normalized relative abundance whereas the smaller fragments
c₁₁, c_8, and c₇ showed an increase in normalized relative abundance. Similar trends were
observed for the doubly- and triply-phosphorylated peptides, see Supplemental Figure 2. For
the doubly phosphorylated peptide (APL_pSFRG_pSLPKSYVK) (Supplementary Figure 2A),
fragments c 14, c 1₃, z 1₃∙, and z 1₂∙ show an overall decrease in normalized relative abundance
with increasing electron energy, whereas c 12, c 11, c 7, and z 11∙ show an increase. The triply
phosphorylated peptide (APL_pSFRG_pSLPK_pSYVK) (Supplementary Figure 2B) shows a
general decrease in normalized relative abundance for the large fragments, c 14, c 1₃, z 1₃∙, z₁2∙,
and z₁1 ∙, and an increase for the fragments c 12 and c7. The results support the hypothesis that
deposition of increasing amounts of energy into the peptide ion enables cleavage of
noncovalent bonds with associated hydrogen rearrangement, i.e., reprotonation of the phospho-
group.

The results above demonstrate that increasing the ECD electron energy improves sequence
coverage for these phosphopeptides. Improved sequence coverage is also obtained when the
triply-charged precursor ions are subjected to ECD. This finding is in agreement with that of
Zubarev et al. [50], i.e., that electron capture cross section, and hence ECD efficiency, increases
with the square of the ion charge. Figure 4 shows the ECD mass spectra of the triply-charged
synthetic peptides acquired at the standard ECD cathode potential (-3.34 V). The ECD mass
spectrum of the unmodified peptide (APLSFRGSLPKSYVK) (Figure 4a) shows cleavage of
11 out of 14 N-Cα bonds, one less than observed for the doubly-charged precursor. Two
doubly-charged fragments (c₁₄²⁺ and c₁₃²⁺) were observed. For the singly-phosphorylated
peptide (APL_pSFRGSLPKSYVK) (Figure 4b), 10 out of 14 N-Cα bonds were cleaved, an
increase of five over the doubly-charged precursor. The site of phosphorylation is
unambiguously identified. Figure 4c shows the ECD mass spectrum of triply-charged peptide
APLSFRG_pSLPKSYVK. Eight of the 14 N-Cα bonds were cleaved. This is not an increase
over the doubly-charged precursor; however the fragments derive from the central region of
the peptide enabling identification of the site of phosphorylation. The ECD mass spectrum of
the triply-charged peptide (APLSFRGSLPK_pSYVK) (Figure 4d) shows cleavage of eight out
of 14 N-Cα bonds, an increase of one cleavage over the doubly-charged precursor. The site of
phosphorylation can be identified from the ECD mass spectrum of the triply-charged ions but
not from the equivalent mass spectrum of the doubly-charged ions. Figure 4e shows the ECD
mass spectrum for the triply-charged doubly-phosphorylated peptide
APL_pSFRG_pSLPKSYVK. Eleven out of 14 N-Cα bonds were cleaved, five more cleavages
than were observed for the doubly-charged ion. One doubly-charged fragment was observed.
The ECD mass spectrum for the triply-charged peptide (APL_pSFRGSLPK_pSYVK) (Figure 4f)
shows 11 N-Cα bonds cleaved, six more than the doubly-charged species. The c ₁₄ ion was the
only doubly-charged fragment. ECD of the final doubly phosphorylated peptide
(APLSFRG_pSLPK_pSYVK) (Figure 4g) resulted in cleavage of 11 N-Cα bonds, four more than
observed for the doubly-charged ion. The ECD mass spectrum of the triply phosphorylated
peptide (APL_pSFRG_pSLPK_pSYVK) (Figure 4h) shows cleavage of 12 of the 14 N-Cα bonds,
seven more than observed for the doubly-charged species. Only one doubly-charged fragment
(c₁₄) was observed.

Unlike the doubly-charged precursors, assigning the charging pattern based on the observed
ECD fragmentation is not straightforward for the triply-charged peptide ions. For example, the
charge on APL_pSFRGSLPKSYVK could arise through deprotonation of the phospho-group
and protonation of the N-terminus, the arginine, and both lysine residues, or simply through
protonation of the basic residues. Contemplation of the fragmentation pattern suggests that in
fact the precursors sample a number of charging patterns, each with an overall charge state of
+3. Similarly, electron capture could occur at the C-terminal lysine residue or at the protonated

Published as: JAm SocMass Spectrom. 2008 September ; 19(9): 1263-1274.

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