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



Creese and Cooper

Page 4


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and β-casein (Sigma Aldrich, Gillingham, UK) were digested with trypsin (Sigma Aldrich)
(1:50, enzyme to protein, wt/wt) in 50 mM ammonium bicarbonate (Sigma Aldrich) (pH 8) at
37 °C overnight. The protein digests were dephosphorylated with calf intestinal alkaline
phosphatase (1 unit per μg protein) (New England Biolabs, Ipswitch, MA) at pH 8 for 1 h at
37 °C. The digests were diluted to a final concentration of ~2 pmol∕μL in methanol:water
(75:25) + 1% formic acid.

ECD-MS/MS

All tandem mass spectrometry analysis was performed on a Thermo Finnegan LTQ FT mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were injected by use of
an Advion Biosciences Triversa electrospray source (Advion Biosciences, Ithaca, NY) at a
flow rate of 200 nL/min.

The mass spectrometer alternated between a full FT-MS survey scan (m/z 400-1800) and 32
subsequent ECD-MS/MS scans of the peptide precursor ions with decreasing cathode potential,
from -1.34 to -16.34 V (0.5 V stepwise) and one ECD scan at cathode potential of 0 V. Survey
scans were acquired in the ICR cell with a resolution of 50,000 at
m/z 400. Precursor ions were
isolated in the ion trap and transferred to the ICR cell for ECD analysis. The isolation width
was 7.5 Th. Automatic gain control was used to accumulate precursor ions in the ion trap (target
5 × 105, maximum fill time 2 s). Electrons for ECD were produced by an indirectly heated
barium-tungsten cylindrical dispenser cathode (5.1 mm diameter, 154 mm from the cell, 1 mm
off axis) (Heat-Wave Labs, Watsonville, CA). The current across the electrode was 1.1 A. Ions
were irradiated with electrons for 70 ms. Each ECD scan consisted of 3 co-added microscans
acquired with resolution of 25,000 at
m/z 400.

Data were analyzed using Xcalibur 2.05 software (Thermo Fisher Scientific). All mass spectra
were manually searched for
a, b, c/c, y, and z∙/z fragment ions.

Results and Discussion

Figure 1 shows the ECD mass spectra of each of the doubly-charged ions of the unmodified
and three singly-phosphorylated synthetic peptides at the “standard” cathode potential (-3.34
V), i.e.; the cathode potential applied in our LC-ECD MS/MS proteomics experiments, and at
the cathode potential that gives the greatest peptide coverage, i.e., the greatest number of N-
Cα cleavages. The ECD mass spectra of the doubly- and triply-phosphorylated peptides are
shown in Supplementary Figure 1, which can be found in the electronic version of this article.
Peptide sequence coverage is summarized in Figure 2. For all phosphopeptides, the maximum
sequence coverage was obtained at ECD cathode potentials between -14 V and -15 V. After
this point, higher mass fragments were not observed, in agreement with the findings of Tsybin
et al. [44]. The ECD mass spectrum obtained for the unmodified peptide under standard
conditions (Figure 1a, left) shows 12 of 14 N-Cα bonds cleaved, the maximum expected due
to the two proline residues present. Additional sequence information is obtained at a cathode
potential of -13.34 V (Figure 1a, right) with the mass spectrum revealing the presence of the
y14 fragment resulting from cleavage N-terminal to Pro2. Presumably the additional electron
energy has enabled this facile cleavage. The ECD mass spectra obtained from the doubly-
charged singly-phosphorylated peptides are shown in Figure 1b, c, and d. For the peptide
(APLpSFRGSLPKSYVK) (Figure 1b, left), five N-Cα bonds are cleaved following ECD at
the standard cathode potential. The
y14 fragment is also observed. The site of phosphorylation
cannot be identified from this mass spectrum. The sequence coverage seen is significantly
worse than that obtained for the unmodified peptide at the same cathode potential: there are
no
z fragments smaller than z12, and no c fragments smaller than c11. The greatest coverage
was achieved with an ECD cathode potential of -14.34 V (Figure 1b, right), 10 of the 14 bonds
N-Cα were cleaved and the site of phosphorylation can be localized. Similar results were

Published as: J Am Soc Mass Spectrom. 2008 September ; 19(9): 1263-1274.



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