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

coulombically). It is postulated that the negatively-charged phosphate group forms a strong
interaction—a salt bridge—with the positively charged lysine side chain [46]. It is well
established that ECD tends not to cleave noncovalent interactions [43]. Any fragments from
the sequence region between the phosphate group and the lysine would not be observed
because, although the peptide backbone has dissociated, the fragments are held together by the
salt bridge. This suggestion is further corroborated by the results obtained for doubly-charged
APLSFRG_pSLPKSYVK (Figure 1c). The fragmentation pattern suggests that the
phosphorylation is deprotonated, both lysines and the arginine are protonated, and that salt
bridge(s) exist between the phospho group and the protonated amino acid side chains. Electron
capture appears to occur at the C-terminal lysine. Again only phosphorylated fragments which
contain arginine and the central lysine are observed. This explanation accounts for the
observation of z 10 and z 11∙ following ECD of [APLSFRG_pSLPKSYVK]²⁺ but not following
ECD of [APLpSFRGSLPKSYVK]²⁺. The ECD of doubly-charged APLSFRGSLPKpSYVK
provides further confirmation—the c₁₁ fragment is not observed (Figure 1d).

The situation is more complicated for the doubly-phosphorylated peptides (Supplementary
Figure 1). If both phospho-groups are deprotonated, the overall charge derives from protonation
of both lysines, the arginine and the N-terminus. The observed fragmentation patterns confirm
this. Only phosphorylated fragments that contain arginine, central lysine, and either the N-
terminus or C-terminal lysine are observed. For example, c₁₁ is observed for
APLpSFRGpSLPKSYVK, but not for APLpSFRGSLPKpSYVK or APLSFRGpSLPKpSYVK.
Fragments z 10 ∙ and z 11 ∙ were observed for APLSFRG_pSLPK_pSYVK but not for either
APL_pSFRG_pSLPKSYVK or APL_pSFRGSLPK_pSYVK. The fragmentation patterns suggest
that multiple noncovalent interactions between deprotonated phospho groups and protonated
amino acid side chains exist. Extension of this hypothesis to the triply-phosphorylated peptide
suggests that all phosphorylations are deprotonated and the N-terminus, both lysines, the
arginine, and another backbone amide nitrogen are protonated. Again, the observed
fragmentation pattern suggests that the phospho-groups are deprotonated and that multiple salt
bridges with protonated amino acid side chains exist.

Increasing the ECD electron energy by decreasing the ECD cathode potential resulted in greater
sequence coverage for all of the phosphopeptides. These results suggest that deposition of
greater amounts of energy on electron capture facilitates cleavage of the noncovalent bonds
and can promote hydrogen rearrangement within the peptide, i.e.; reprotonation of the phospho-
groups. In addition, cleavage of the noncovalent bonds increases conformational heterogeneity,
which plays a role in increased peptide sequence coverage [47, 48]. For peptide
APL_pSFRGSLPKSYVK, increasing the ECD electron energy resulted in the appearance of
fragments z7, z9, z 10, z 11 ∙, and c6, c7, c8, c 10. The appearance of z₇ and z₉ can be explained
purely on the basis of cleavage of the salt bridge. Formation of the remaining fragments,
however, must involve hydrogen rearrangement. Without that, z₁₀, and z₁₁∙ would be doubly-
charged and c₆, c₇, c₈, c₁₀ would be neutral. Similarly, for peptide APSFRG_pSLPKSYVK, the
appearance of z₇ and c₆, c₇ can be explained by straightforward cleavage of the salt bridge,
however c₈ and c₁₀ require reprotonation of the phospho-group. Figure 3 shows the normalized
relative abundance of the major ECD fragments from doubly-charged precursors of unmodified
APLSFRGSLPKSYVK (Figure 3a) and singly-phosphorylated APLSFRGSLPKpSYVK
(Figure 3b). Only fragments detected in four or more successive scans are included. The relative
abundance was calculated as the ratio of the intensity of the fragment to the sum of the
intensities of all the fragments (excluding the charge-reduced species). The unmodified peptide
(APLSFRGSLPKSYVK) (Figure 3a) did not show marked differences in the normalized
relative abundances of the fragments: Fragments c₁₁, z₁₃∙, z₁₂∙, z₁₁∙, and z₁₀∙ showed slight
decreases, fragments c₁₂, c₁₀, c₈, and c₇ showed slight increases and fragments c₁₄ and c₁₃ did
not vary with decreasing ECD cathode potential (increasing electron energy). Tsybin et al.
[49] have shown previously that increasing electron energy correlates with a reduction in

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

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