Top-Down Mass Analysis of Protein Tyrosine Nitration: Comparison of Electron Capture Dissociation with “Slow-Heating” Tandem Mass Spectrometry Methods

Table 1. Numbers of MS/MS Fragments from ECD, IRMPD, and CID of Nitrated Myoglobin^a

^a nY103 indicates nitrotyrosine.

Wave Laboratories, Watsonville, California). The current
across the electrode was ~1.1 A. The ECD duration was
5-10 ms, and the ECD energy was in the range of 2.5-4%
(corresponding to a cathode potential of -0.275 to -1.775
V). IRMPD of the protein ions was carried out in the ICR
cell using a 75 W in-built CO2 laser (Synrad, Mikilteco,
Washington) for 100 ms. IRMPD energy was measured as
a percent of the maximum (i.e., 75 W). Raw MS data were
analyzed by use of Xcalibur 2.05 software (Thermo Fisher
Scientific), where the Xtract program was used for calculat-
ing monoisotopic masses (44% fit factor, 25% remainder).
ProSight PTM (https://prosightptm.scs.uiuc.edu) was used
to search for b, y and c, z protein fragment ions in the single
protein mode. The mass accuracy for the search was set at
10 ppm.

RESULTS

Nitrated Myoglobin. Electrospray mass spectrometry of the
nitrated myoglobin indicated that both unmodified and mono-
nitrated proteins with relative ratio of 1:1.25 were present in
the sample. Nitration of myoglobin was expected to take place
at Tyr103.⁴² MS/MS of nitrated myoglobin ions was carried
out for five charge states: 14+, 16+, 18+, 20+, and 22+, Table
1. For each charge state the total number of fragments from
ECD was greater than that from IRMPD or CID. For each MS/
MS method, the number of fragments varied with the charge
state but not monotonically. MS/MS spectra and fragmentation
diagrams for the 20+ ions of nitrated myoglobin are given in
Figure 1. MS/MS fragmentation diagrams for the 20+ ions of
unmodified myoglobin are given in Figure S-1 in the Supporting
Information. ECD of the unmodified myoglobin produced
extensive cleavages, while IRMPD and CID resulted in smaller
numbers of fragments. CID of unmodified myoglobin produced
more fragments than IRMPD (35 versus 26). Nitration resulted
in a reduction of the total number of ECD fragments (from 84
to 45), and ECD cleavage sites were located at least 5 residues
C-terminal and 11 N-terminal to the site of nitration, Tyr103,
versus two and five residues, respectively, in the unmodified
protein, Figure 1a. A decrease in the number of ECD cleavages
around Tyr103 upon nitration was observed for all charge states.
IRMPD of the nitrated myoglobin produced cleavages of the
protein backbone closer to the site of nitration than ECD,
Figure 1c. IRMPD produces more fragments than CID from
the nitrated protein (25 vs 12), Figure 1c,d. Nitration does not
seem to have any serious effect on the efficiency of IRMPD
(26 and 25 IRMPD fragments produced from unmodified and
nitrated myoglobin, respectively). The number of CID frag-

7286 Analytical Chemistry, Vol. 82, No. 17, September 1, 2010
ments reduced significantly after nitration (35 and 12 fragments
before and after nitration, respectively). The reason for that
appears to be of the instrumental nature (see the discussion
below). Additionally, our ECD, CID, and IRMPD results all
confirm unequivocally that Tyr103, not Tyr146, is the site of
nitration. However, fewer fragments confirming nitration on
Tyr103 were produced by CID of the 20+ ions than by ECD or
IRMPD. The same was true for the other charge states.

Nitrated Lysozyme. Electrospray mass spectrometry of the
two LC fractions from lysozyme nitration indicated that one of
them contained mostly mononitrated protein and the other
mostly bis-nitrated protein. MS/MS of unmodified, mono, and
bis-nitrated lysozyme was carried out for both nonreduced and
reduced protein samples. Initial nitration of lysozyme was
expected to take place at Tyr23 followed by nitration of
Tyr20.^43,44 MS/MS spectra and fragmentation diagrams for the
most abundant charge state, 15+, of reduced bis-nitrated
lysozyme are presented in Figure 2. Summaries of fragments
from ECD MS/MS of the 10+ ions of nonreduced lysozyme
are presented in Figure S-2 in the Supporting Information.
Higher charge states of nonreduced lysozyme could not be
obtained with abundances sufficient for MS/MS investigation.
MS/MS spectra and fragmentation diagrams for the 15+ charge
state of reduced mononitrated lysozyme are presented in
Figures S-3-S-5 in the Supporting Information.

Only small numbers of fragments could be obtained from ECD
of nonreduced lysozyme, Figure S-2 in the Supporting Information.
As the cleavage of a disulfide bond by ECD results in one of the
two cysteines reduced by the recombined proton,²⁴ the search
for ECD fragments from the nonreduced lysozyme was carried
out assuming a -1 Da mass shift on one of the cysteines from
each of the four S-S bonds, no mass shift on the other cysteine,
and vice versa. However, the fragments found indicate that only
the Cys6-Cys127 disulfide bond was cleaved by ECD, with Cys6
being reduced. The other disulfide bonds appear not to be cleaved
by ECD. A total of 13 c' fragments between amino acid residues
3 and 20 and only two z∙ fragments close to the C-terminus were
produced from unmodified native lysozyme, Figure S-2a in the
Supporting Information. ECD of mono- or bis-nitrated nonre-
duced lysozyme produces fewer fragments than ECD of unmodi-
fied nonreduced lysozyme, Figure S-2b,c in the Supporting
Information. Furthermore, ECD does not cleave beyond amino
acid residue 13. Thus ECD cleavages do not reach the site(s) of
nitration, and none of the ECD fragments contains the site(s) of
nitration. Use of activated ion (AI) ECD, as described in our recent

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