Creese and Cooper
Page 5
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obtained for peptide APLSFRGpSLPKSYVK (Figure 1c). At the standard ECD cathode
potential eight N-Cα bonds were cleaved. The site of phosphorylation can be identified by
elimination, however no fragments adjacent to the site of modification were observed. No c
ions smaller than c11 and no z ions smaller than z10 were observed. An ECD cathode potential
of -14.34 V (Figure 1c, right) gave the greatest peptide coverage with 11 out of 14 N-Cα bonds
cleaved. Fragments were observed adjacent to the site of phosphorylation. For peptide
APLSFRGSLPKpSYVK (Figure 1d), 7 of the 14 N-Ca bonds were cleaved at the standard
cathode potential. No c fragment ions smaller than c12 and no z ions smaller than z10 were
identified. The site of phosphorylation could not be identified from this spectrum. The best
coverage was achieved with an ECD cathode potential of -14.84 V (Figure 1d, right): 10 out
of 14 N-Ca bonds were cleaved, in addition to production of the a9∙ and y 14 fragments. The
site of phosphorylation can be identified.
The ECD mass spectra of the multiply-phosphorylated peptides show the same trend as those
of the singly-phosphorylated peptides (see Figure 2). The ECD mass spectrum of the doubly
phosphorylated peptide (APLpSFRGpSLPKSYVK) (Supplementary Figure 1) shows cleavage
of 6 of 14 N-Ca bonds. The sites of phosphorylation may be identified by elimination but there
are no c fragments smaller than c 11, and no z fragments smaller than z 12∙. Eleven out of fourteen
backbone bonds were cleaved when the cathode potential is increased to -14.84 V. Ten of
these result in c and z ∙ ions and one of the cleavages produces a y ion. Note that the
phosphorylation is retained on y12. The ECD mass spectrum of the peptide
APLpSFRGSLPKpSYVK (Supplementary Figure 1B) obtained with standard cathode
potential shows cleavage of 5 out of 14 N-Ca bonds, plus the y12 fragment ion. The sites of
phosphorylation cannot be localized. The greatest coverage was achieved with an ECD cathode
potential of -14.84 V (Supplementary Figure 1B, right): 10 of 14 N-Ca bonds were cleaved
in addition to production of three y and three a ∙ fragment ions. Both phosphorylation sites are
identified. The ECD mass spectrum of the doubly-phosphorylated peptide
APLSFRGpSLPKpSYVK (Supplementary Figure 1C) shows 7 of 14 N-Ca bonds were cleaved
following ECD with standard cathode potential. The sites of phosphorylation may be identified
by elimination; however, no fragments adjacent to the modification sites were observed. The
greatest sequence coverage is achieved with an ECD cathode potential of -14.84 V: 9 of the
14 N-Ca bonds are cleaved. Both sites of modification are unambiguously identified. The ECD
mass spectrum of the triply-phosphorylated peptide APLpSFRGpSLPKpSYVK
(Supplementary Figure 1D) obtained with a standard cathode potential reveals cleavage of five
of the 14 N-Ca bonds. The greatest coverage was achieved with an ECD cathode potential of
-14.34 V. Eleven of the 14 N-Ca bonds were cleaved.
The results obtained for the doubly-charged singly-phosphorylated peptides can be explained
by consideration of the charging pattern and the presence of noncovalent interactions in the
form of salt bridges. In the unmodified peptide ion, the charge arises through protonation of
the arginine residue and the C-terminal lysine. The arginine is the most basic amino acid residue
and the C-terminal lysine the farthest site from the arginine. Electron capture occurs at the least
basic site [45], i.e., the lysine. This is confirmed by the fragmentation pattern (Figure 1a)
observed for the unmodified peptide: All the observed fragments contain the (protonated)
arginine residue. The fragmentation pattern observed for doubly-charged
APLpSFRGSLPKSYVK suggests that the phosphorylation is deprotonated and that the overall
2+ charge is achieved through protonation of the arginine and both lysines. Electron capture
appears to occur at the C-terminal lysine and only phosphorylated fragments which contain
both the arginine and the central lysine are observed (Figure 1b). However, the results cannot
be explained by the charging pattern alone. For example, one might expect to observe fragments
z5 and z7 through z9 if the central lysine was protonated. Moreover, doubly-charged z10 and
z11 fragments might be observed and this was not the case. (Note, however, that it is likely any
doubly-charged fragments and their negatively charged complements would be held together
Published as: J Am Soc Mass Spectrom. 2008 September ; 19(9): 1263-1274.