Protein Protocols Protein Protocols

Mass Spectrometry of Protein Phosphorylation 617
3.7. Localization of Sites of Phosphorylation by LC/MS/MS
After identifying a candidate ion from the initial LC/MS analysis, the sample is
applied to LC and the ion is selected for on-line collision-induced dissociation (CID)
to determine the site of phosphorylation. Peptides usually fragment at peptide bonds to
produce a series of daughter ions containing the N-terminal or C-terminal ends of the
molecule (“b” ions or “y” ions, respectively) allowing the sequence to be “read” from
the mass differences between the fragment ions. For example, Fig. 4 shows a comparison
of the MS/MS spectra of unphosphorylated and phosphorylated CP4 peptide derived
from the head domain of the interphase form of Drosophila melanogaster lamin (17).
The peptide with the sequence PPSAGPQPPPPSTHSQTASSPLSPTR has nine potential
phosphorylation sites. An identical set of unphosphorylated fragment ions, whose
masses correspond to those predicted for y19-y6 (Fig. 4A,B) was obtained. This places
the site of phosphorylation beyond y19. As there is only one candidate serine beyond
y19, it can be concluded that the serine at position three (which corresponds to Ser25 in
the lamin protein) is the site of phosphorylation. Positive identification is seen in a set
of ions, corresponding to b2–b7 where b3–b7 is shifted by 80 Da in the phospho-CP4 MS/MS spectrum compared with the nonphosphorylated CP4 peptide. Also note the
loss of phosphoric acid, giving rise to an intense signal 27 Da lower than the triply
charged precursor ion (Fig. 4B).
Figure 2 demonstrates that the phosphorylation of K2 isolated from M-phase lamin
was increased when compared with the same fragment isolated from interphase lamin.
This could mean either that the previously identified serine at position 3 (corresponding
to Ser25 in the lamin protein) becomes phosphorylated to a greater extent than in
interphase lamin, or that a site other than Ser25 is phosphorylated to higher stoichiometry.
These two possibilities can be distinguished by subjecting the same peptide isolated
from interphase and M-phase lamin to fragmentation. 80 Da shifts due to the presence
of a phosphorylated residue can be observed for y10–y18 in the spectrum of phospho-
CP4 derived from M-phase lamin, as well as fragments corresponding to y6–y13 after
neutral loss (Fig. 5A). This indicates that the site of phosphorylation of the head region
in M-phase lamin has changed to a residue located C-terminally of Ser25. Identification of
the phosphorylated residue in the M-phase CP4 fragment becomes evident upon
inspection of the low mass range of the spectrum. In both phosphopeptides (interphase
and M-phase forms) the y3 ions are identical (compare Figs. 4 and 5). This eliminates
the Thr47 as the phosphorylated site. As neutral loss of phosphate occurs from y6 on
in the M-phase CP4 peptide, and because there is only one serine residue between
y3 and y6, it can be concluded that Ser45 is the residue that becomes phosphorylated in
M-phase lamin (Fig. 5). Therefore, during embryonal development of the fruit fly,
phosphorylation of the head domain of lamin is regulated by changing the site of
phosphorylation from Ser45 in early embryos to Ser25 in older embryos. This leads
to profound changes in the ability of lamin to self polymerize: whereas lamin
phosphorylated on Ser45 is soluble, lamin phosphorylated on Ser25 polymerizes to form
the underlying structure of the nuclear envelope. This example demonstrates the use of
mass spectrometry to trace protein phosphorylation in organisms that are not amenable
to classical phosphoprotein mapping by metabolic 32P-labeling.